Molecular linkers suitable for crystallization and structural analysis of molecules of interest, method of using same, and methods of purifying g protein-coupled receptors

ABSTRACT

A method of crystallizing a molecule-of-interest is disclosed. The method comprises (a) contacting molecules of the molecule-of-interest with at least one type of heterologous molecular linker being capable of interlinking at least two molecules of said molecule-of-interest to thereby form a crystallizable molecular complex of defined geometry; and (b) subjecting said crystallizable molecular complex to crystallization-inducing conditions, thereby generating the crystal containing said molecule-of-interest.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to molecular linkers suitable forcrystallization and structural analysis of molecules of interest, tomethod of using same, and to methods of purifying G protein coupledreceptors (GPCRs). More particularly, the present invention relates tomethods of crystallizing membrane proteins and to methods of purifyingGPCRs via affinity chromatography using arrestin derived polypeptides.

Importance of protein structure determination: The recently fullysequenced human genome, has been found to contain up to 38,000 genes(Venter J C. et al., 2001. Science 291:1304) encoding up to an order ofmagnitude more protein species. It is evident that the informationcontained therein holds tremendous potential for furthering thedevelopment of practical applications in all fields involving the lifesciences. However, most proteins remain to be characterized with respectto their structure and function and, although the transcription profilesof the genes encoding these proteins are currently being determined,such data can yield only limited information. In order to fully harnessthe potential of the information contained in the complete human genomesequence, it will be necessary to systematically determine thethree-dimensional (3D) structure of the proteins encoded therein.

The capacity to solve the 3D atomic structure of proteins is proving tobe crucial for understanding and regulating their biological functionsand, as such, is playing an increasingly vital role in the advancementof biomedical science and biotechnology, in particular in the realm ofdrug design.

The pathogenesis of a very large number of human diseases involvesmembrane proteins such as GPCRs, as startlingly demonstrated by the factthat a 60% majority of approved drugs elicit their therapeutic effectsby selectively targeting members of the GPCR family (GlaxoWellcome,1996. Nature Suppl. 384:1-5). However, pharmacological treatment ofdiseases involving GPCRs remains far from optimal and there is thus acritical need for novel and improved GPCR-targeting drugs. Ashighlighted, for example, by the 3D atomic structure-based developmentof protease inhibitors employed in the first effective treatment ofhuman immunodeficiency virus (HIV) induced acquired immuno-deficiencysyndrome (AIDS) (Wlodawer A. and Vondrasek J., 1998. Annu Rev BiophysBiomol Struct. 27:249), the development of novel and improved membraneprotein-targeting drugs, such as GPCR-targeting drugs, can dramaticallybenefit from the availability of the 3D atomic structure of such drugtargets.

Other increasingly important applications of protein crystals includetheir use as catalysts on a commercial scale, in bioremediation andgreen chemistry applications, and in purification-related applications,such as enantioselective chromatography of pharmaceuticals andhigh-grade chemicals. In the near future, their utility will furtherexpand to include the purification of protein drugs and the developmentof adjuvant-less vaccines (Margolin A L. and Navia M A., 2001.Angewandte Chemie International Edition 40:2204).

General obstacles to protein crystallization: The bottleneck indetermination of novel protein structures has shifted from thecollection and interpretation of crystallographic data to the productionof large amounts of highly pure protein and the generation ofdiffraction-grade crystals. Techniques for growing such crystalscurrently rely substantially on empirical processes for which onlygeneral rules of thumb are available and which frequently requireadaptations tailored to accommodate the peculiarities of individualproteins.

Several factors contribute to the difficulty in obtaining highly orderedprotein crystals. Although contacts between crystallized proteinmolecules are of comparable energy to those between small molecules, thesignificantly fewer number of intermolecular contacts per molecularweight of crystallized protein molecules renders these contacts veryfragile (Carugo O. and Argos P., 1997. Protein Science 6:2261).Furthermore, due to their inherent complexity, protein molecules canassume numerous conformations, a phenomenon which tends to preventformation of highly ordered crystals. Moreover, aggregated proteins areable to form many different types of intermolecular contacts of whichonly a restricted number will generate highly ordered crystals. Hence,crystallization conditions must be carefully fine-tuned so as to inducethe proper molecular conformation and packing orientation of eachmolecule accreted during the process of crystallization. Such conditionsare difficult to obtain since small variations in physico-chemicalparameters, such as pH, ionic strength, temperature or contaminants,will strongly influence the process of crystallization in a way that isunique for each protein due to the diversity of the chemical groups andpossible configurations thereof involved in the formation ofintermolecular contacts (Giege R. et al., Acta Crystallographica SectionD-Biological Crystallography 1994. 50:339; Durbin S D. and Feher G.,1996. Annu Rev Phys Chem. 47:171; Weber P C., Overview of proteincrystallization methods, in Macromolecular Crystallography, Pt a. 1997.p. 13-22; Chernov A A., Physics Reports-Review Section of PhysicsLetters 1997. 288:61; Rosenberger F., Theoretical and TechnologicalAspects of Crystal Growth 1998. p. 241; Wiencek J M., 1999. Annu RevBiomed Eng. 1:505).

Obstacles to Membrane Protein Crystallization

Three dimensional protein structure determination at high resolutionrepresents a particularly difficult challenge for membrane proteins andthe number of such proteins that have been crystallized is still smalland far behind that of soluble proteins, even though membrane proteinsrepresent up to 40% of the proteins encoded by the human genome (WallinE. and von Heijne G., 1998. Protein Sci. 7:1029).

The crystallization of membrane proteins is particularly difficult dueto the fact that, unlike soluble proteins which tend to have hydrophilicsurfaces and polar cores, membrane proteins have significant hydrophobicsurfaces through which they interact with membrane lipids. Such proteinsexist in a quasi-solid state in the membrane and are not readily solublein either aqueous or apolar environments.

The most widely employed approach for solubilization of membraneproteins is the use of detergents interacting with the hydrophobicsurfaces of the protein to generate mixed detergent/protein micelles.Solubilized membrane proteins can then be crystallized in an orderedtwo-dimensional (2D) lattice by reconstitution in an artificial lipidbilayer, allowing 2D structural determination via electron microscopy.While such 2D crystals are relatively easy to obtain, the use ofelectron microscopy for determining molecular structure suffers from thesignificant drawback of generating structural information with poorresolution in directions orthogonal to the 2D lattice, thus preventingstructural determination at sufficiently high resolutions (Stowell M H.et al., 1998. Curr Opin Struct Biol. 8:595). An additional factorcontributing to the difficulty of determining the structure of membraneproteins at high resolution is due to the fact that crystal contactsmade between detergent micelles tend to be disordered, resulting inpoorly diffracting crystals. Although the use of helical crystals andadvanced image processing can obviate some of these drawbacks, it isonly with X-ray crystallography of 3D crystals that high resolutiondetermination of 3D protein structure can be achieved. This isessential, for example, to generate detailed pictures of moleculartarget sites when designing drugs specifically interacting with suchsites. In the case of membrane proteins, this is highly desirable sincesuch information can significantly contribute to the design anddevelopment of novel drugs for the very large number of diseases whosepathogenesis involves membrane proteins, such as receptors. Suchdiseases include, for example, cancer, viral diseases such as AIDS,neurological disorders, metabolic illnesses such as diabetes, etc.

Prior Art Optimization of Crystallization Conditions

High Throughput Techniques

High throughput techniques are currently being employed to determine theconditions required for growth of protein crystals. One such approachemploys automation to perform large numbers of crystallization trials(Morris, D W. et al., 1989. Biotechniques 7:522; Zuk W M. and Ward K B.,1991. Journal of Crystal Growth 110:148; Heinemann U. et al., 2000.Progress in Biophysics & Molecular Biology 73:347).

Such high throughput approaches employ the sparse-matrix proteincrystallization method, in which a series of crystallization conditionsare tested in parallel, the most promising ones being iterativelyrefined until crystallization is achieved (Jancarik J. and Kim S H.,1991. Journal of Applied Crystallography 24:409; Cudney B., et al.,1994. Acta Crystallographica Section D-Biological Crystallography50:414; Hennessy D. et al., 2000. Acta Crystallographica SectionD—Biological Crystallography 56:817).

However, successful crystallization of membrane proteins via suchtechniques is highly inefficient due to the high tendency of membraneproteins to denature and/or aggregate during crystallization.Furthermore, such methods, being substantially empirical, present thedisadvantages of being both time-consuming and of requiring largeamounts of pure protein, a requirement which is generally difficult orexpensive to fulfill.

One strategy which has been suggested in order to circumvent thedisadvantages inherent to such high throughput techniques is to assistthe crystallization of molecules which are otherwise difficult orimpossible to crystallize by either modifying such molecules so as tofacilitate their crystallization, or by crystallizing such molecules incomplex with other molecules susceptible to provide an ordered matrixfacilitating formation of the basic unit of a crystal lattice.

Protein-modification techniques: One approach attempting to improvemembrane protein crystal growth and ordering has employed complexationof a protein of interest with antibody fragments prior tocrystallization (Hunte C., 2001. FEBS Lett. 504:126-32; Lange C. & HunteC., 2002. Proc Natl Acad Sci USA. 99:2800-5; Ostermeier C. and MichelH., 1997. Curr Opin Struct Biol. 7:697; Ostermeier C. et al., 1997. ProcNatl Acad Sci USA. 94:10547-53).

Another modification based approach has used fusion of proteins to becrystallized to large hydrophobic domains derived from heterologousproteins in an attempt to minimize the overall hydrophobicity ofproteins of interest (Prive G. et al., 1994. Biol Crystallogr. D50:375).

Yet another approach involves alteration and engineering of crystal unitcell contacts, an example being the crystallization of apoferritin bysite-directed mutagenesis of residues involved in the binding of a Co²⁺atom introduced during the crystallization process (Takeda S. et al.,1995. Proteins, 23:548).

These approaches, however, have the significant drawback thatidentifying and creating suitable fusion proteins or engineeringresidues involved in crystal contacts are ad hoc and very laborintensive procedures requiring much fine tuning for applicability to anygiven protein.

Functionalized lipids: Still another approach has employed binding offunctionalized lipids to proteins of interest in an attempt to generatecrystalline arrays of such proteins. For example, divalent metalion-chelated lipids or electrostatically charged lipids have beenemployed to bind proteins via specific surface histidine residues or viacomplementarily charged residues, respectively. The use of planar layersof such lipids has been employed to generate 2D crystals (Frey W. etal., Proc Nat Acad Sci. USA 1996 93:4937) which can be studied byelectron microscopy, but not by X-ray diffraction, thereby yieldinglimited structural information in terms of dimensionality and in termsof resolution.

A more advanced variant of this approach has utilized lipid nanotubes togenerate helical crystals (Wilson-Kubalek, E. et al., Proc. Natl. Acad.Sci. U.S.A. 1998, 95:8040). These crystals, however, can only be used todetermine 3D protein structure at low resolution using electronmicroscopy and thus cannot be employed to solve molecular structure atatomic resolution, as is the case with X-ray crystallography.

Thus, all prior art approaches have failed to provide an adequatesolution for efficiently generating X-ray diffraction grade crystals ofmolecules such as membrane proteins.

There is thus a widely recognized need for and it would be highlyadvantageous to have, a method of crystallizing molecules, such asmembrane proteins, devoid of the above limitations.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of generating a crystal containing a molecule-of-interest, themethod comprising: (a) contacting molecules of the molecule-of-interestwith at least one type of heterologous molecular linker being capable ofinterlinking at least two molecules of the molecule-of-interest tothereby form a crystallizable molecular complex of defined geometry; and(b) subjecting the crystallizable molecular complex tocrystallization-inducing conditions, thereby generating the crystalcontaining the molecule-of-interest.

According to further features in preferred embodiments of the inventiondescribed below, the at least one type of heterologous molecular linkeris selected such that the crystallizable molecular complex formed iscapable of generating a crystal selected from the group consisting of a2D crystal, a helical crystal and a 3D crystal.

According to still further features in preferred embodiments, themolecule-of-interest is a polypeptide.

According to still further features in preferred embodiments, thepolypeptide is a membrane protein.

According to still further features in preferred embodiments, themembrane protein is a G protein coupled receptor.

According to still further features in preferred embodiments, the Gprotein coupled receptor is rhodopsin or is a class A G protein coupledreceptor.

According to still further features in preferred embodiments, the classA G protein coupled receptor is m2 muscarinic cholinergic receptor.

According to still further features in preferred embodiments, the atleast one type of heterologous molecular linker includes a region forspecifically binding the molecule-of-interest.

According to still further features in preferred embodiments, themolecule-of-interest is a G protein coupled receptor and the region forspecifically binding the molecule-of-interest comprises a moleculeselected from the group consisting of at least a portion of an arrestinmolecule, at least a portion of an arrestin molecule having a mutationat an amino acid residue position corresponding to position 90 in bovinevisual arrestin, at least a portion of an arrestin molecule having amutation at an amino acid residue position corresponding to position 175in bovine visual arrestin, SEQ ID NO: 3, and SEQ ID NO: 4.

According to still further features in preferred embodiments, the atleast a portion of an arrestin molecule is homologous to amino acidresidues 11 to 190, or 11 to 370 of human beta-arrestin-1a.

According to still further features in preferred embodiments, the atleast a portion of an arrestin molecule comprises a G protein coupledreceptor-binding domain of the arrestin molecule.

According to still further features in preferred embodiments, themutation at an amino acid residue position corresponding to position 90in bovine visual arrestin is a mutation to a serine or threonineresidue.

According to still further features in preferred embodiments, themutation at an amino acid residue position corresponding to position 175in bovine visual arrestin is a mutation to a glutamic acid or anasparagine residue.

According to still further features in preferred embodiments, the Gprotein coupled receptor is rhodopsin or is a class A G protein coupledreceptor.

According to still further features in preferred embodiments, the classA G protein coupled receptor is m2 muscarinic cholinergic receptor.

According to still further features in preferred embodiments, themolecule-of-interest includes a histidine tag and the region forspecifically binding the molecule-of-interest comprises a nickel ion oran antibody specific for the histidine tag.

According to still further features in preferred embodiments, themolecule-of-interest includes core streptavidin and the region forspecifically binding the molecule-of-interest comprises a biotin moietyor a Strep-tag.

According to still further features in preferred embodiments, themolecule-of-interest includes a biotin moiety or a Strep-tag and theregion for specifically binding the molecule-of-interest comprises corestreptavidin.

According to still further features in preferred embodiments, themolecule-of-interest is a G protein coupled receptor and the at leastone type of molecular linker comprises a molecule selected from thegroup consisting of at least a portion of an arrestin molecule, at leasta portion of an arrestin molecule having a mutation at an amino acidresidue position corresponding to position 90 in bovine visual arrestin,at least a portion of an arrestin molecule having a mutation at an aminoacid residue position corresponding to position 175 in bovine visualarrestin, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

According to still further features in preferred embodiments, the atleast a portion of an arrestin molecule is homologous to amino acidresidues 11 to 190, or 11 to 370 of human beta-arrestin-1a.

According to still further features in preferred embodiments, the atleast a portion of an arrestin molecule comprises a G protein coupledreceptor-binding domain of the arrestin molecule.

According to still further features in preferred embodiments, themutation at an amino acid residue position corresponding to position 90in bovine visual arrestin is a mutation to a serine or threonineresidue.

According to still further features in preferred embodiments, themutation at an amino acid residue position corresponding to position 175in bovine visual arrestin is a mutation to a glutamic acid or anasparagine residue.

According to still further features in preferred embodiments, the Gprotein coupled receptor is rhodopsin or is a class A G protein coupledreceptor.

According to still further features in preferred embodiments, the classA G protein coupled receptor is m2 muscarinic cholinergic receptor.

According to still further features in preferred embodiments, the atleast one type of heterologous molecular linker includes at least twonon-covalently bound subunits.

According to still further features in preferred embodiments, the atleast two non-covalently bound subunits comprise a first subunitcomprising a homomultimerizing portion and a metal-binding portion, anda second subunit comprising a portion specifically binding themolecule-of-interest, According to still further features in preferredembodiments, the at least two non-covalently bound subunits comprise afirst subunit comprising a homomultimerizing portion and a portionspecifically binding the molecule-of-interest, and a second subunitcomprising a metal-binding portion, and a portion specifically bindingthe first subunit.

According to still further features in preferred embodiments, the atleast one type of heterologous molecular linker includes a moleculeselected from the group consisting of a polycyclic molecule, apolydentate ligand, a macrobicyclic cryptand, a polypeptide and a metal.

According to still further features in preferred embodiments, the atleast one type of heterologous molecular linker comprises corestreptavidin.

According to still further features in preferred embodiments, the atleast one type of heterologous molecular linker is selected so as todefine the spatial positioning and orientation of the at least twomolecules within the crystallizable molecular complex, therebyfacilitating crystallization of the molecule-of-interest.

According to still further features in preferred embodiments, the atleast one type of heterologous molecular linker includes a hydrophilicregion, the hydrophilic region being for facilitating crystallization ofthe molecule-of-interest.

According to still further features in preferred embodiments, the atleast one type of heterologous molecular linker includes aconformationally rigid region, the conformationally rigid region beingfor facilitating crystallization of the molecule-of-interest.

According to still further features in preferred embodiments, the atleast one type of heterologous molecular linker includes a metal-bindingmoiety capable of specifically binding a metal atom, the metal atombeing capable of facilitating crystallographic analysis of the crystal.

According to still further features in preferred embodiments, themetal-binding moiety is a metal binding protein.

According to still further features in preferred embodiments, the metalbinding protein is metallothionein.

According to still further features in preferred embodiments, the atleast one type of heterologous molecular linker includes a region beingcapable of functioning as a purification tag, the purification tag beingcapable of facilitating purification of the crystallizable molecularcomplex and/or of facilitating the interlinking at least two moleculesof the molecule-of-interest.

According to still further features in preferred embodiments, the regionbeing capable of functioning as a purification tag is selected from thegroup consisting of a T7 tag, a histidine tag, a Strep-tag, corestreptavidin, and biotin.

According to still further features in preferred embodiments, themolecule-of-interest includes a region being capable of functioning as apurification tag, the purification tag being capable of facilitatingpurification of the crystallizable molecular complex, and/or offacilitating the interlinking at least two molecules of themolecule-of-interest.

According to still further features in preferred embodiments, the regionbeing capable of functioning as a purification tag is selected from thegroup consisting of a T7 tag, a histidine tag, a Strep-tag, corestreptavidin, and biotin.

According to still further features in preferred embodiments, themolecule-of-interest includes a metal-binding moiety capable ofspecifically binding a metal atom, the metal atom being capable offacilitating crystallographic analysis of the crystal.

According to still further features in preferred embodiments, themetal-binding moiety is a metal binding protein.

According to still further features in preferred embodiments, the metalbinding protein is metallothionein.

According to another aspect of the present invention there is provided amethod of generating a crystal containing a polypeptide of interest, themethod comprising: (a) providing a molecule including the polypeptide ofinterest and a heterologous multimerization domain being capable ofdirecting the homomultimerization of the polypeptide of interest; (b)subjecting the molecule to homomultimerization-inducing conditions,thereby forming a crystallizable molecular complex; and (c) subjectingthe crystallizable molecular complex to crystallization-inducingconditions, thereby generating the crystal containing the polypeptide ofinterest.

According to further features in preferred embodiments of the inventiondescribed below, steps (a) and (b) are effected concomitantly.

According to still further features in preferred embodiments, theheterologous multimerization domain is selected such that thecrystallizable molecular complex formed is capable of generating acrystal selected from the group consisting of a 2D crystal, a helicalcrystal and a 3D crystal.

According to still further features in preferred embodiments, theheterologous multimerization domain includes a hydrophilic region, thehydrophilic region being for facilitating crystallization of thepolypeptide of interest.

According to still further features in preferred embodiments, theheterologous multimerization domain includes a conformationally rigidregion, the conformationally rigid region being for facilitatingcrystallization of the polypeptide of interest.

According to still further features in preferred embodiments, theheterologous multimerization domain is selected so as to define thespatial positioning and orientation of polypeptides of the polypeptideof interest within the crystallizable molecular complex, therebyfacilitating crystallization of the polypeptide of interest.

According to still further features in preferred embodiments, theheterologous multimerization domain comprises core streptavidin.

According to still further features in preferred embodiments, thepolypeptide of interest is a G protein coupled receptor and theheterologous multimerization domain comprises a molecule selected fromthe group consisting of at least a portion of an arrestin molecule, atleast a portion of an arrestin molecule having a mutation at an aminoacid residue position corresponding to position 90 in bovine visualarrestin, at least a portion of an arrestin molecule having a mutationat an amino acid residue position corresponding to position 175 inbovine visual arrestin, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, andSEQ ID NO: 6.

According to still further features in preferred embodiments, the atleast a portion of an arrestin molecule is homologous to amino acidresidues 11 to 190, or 11 to 370 of human beta-arrestin-1a.

According to still further features in preferred embodiments, the atleast a portion of an arrestin molecule comprises a G protein coupledreceptor-binding domain of the arrestin molecule.

According to still further features in preferred embodiments, themutation at an amino acid residue position corresponding to position 90in bovine visual arrestin is a mutation to a serine or threonineresidue.

According to still further features in preferred embodiments, themutation at an amino acid residue position corresponding to position 175in bovine visual arrestin is a mutation to a glutamic acid or anasparagine residue.

According to still further features in preferred embodiments, the Gprotein coupled receptor is rhodopsin or is a class A G protein coupledreceptor.

According to still further features in preferred embodiments, the classA G protein coupled receptor is m2 muscarinic cholinergic receptor.

According to still further features in preferred embodiments, thepolypeptide of interest includes a histidine tag and the heterologousmultimerization domain comprises a nickel ion or an antibody specificfor the histidine tag.

According to still further features in preferred embodiments, thepolypeptide of interest includes core streptavidin and the heterologousmultimerization domain comprises a biotin moiety or a Strep-tag.

According to still further features in preferred embodiments, thepolypeptide of interest includes a biotin moiety or a Strep-tag and theheterologous multimerization domain comprises core streptavidin.

According to still further features in preferred embodiments, thepolypeptide of interest and the heterologous multimerization domain areinterlinked via a molecular linker.

According to still further features in preferred embodiments, at leastone of the heterologous multimerization domain and the molecular linkerinclude a hydrophilic region, the hydrophilic region being forfacilitating crystallization of the polypeptide of interest.

According to still further features in preferred embodiments, at leastone of the heterologous multimerization domain and the molecular linkerinclude a conformationally rigid region, the conformationally rigidregion being for facilitating crystallization of the polypeptide ofinterest.

According to still further features in preferred embodiments, at leastone of the heterologous multimerization domain and the molecular linkeris selected so as to define the spatial positioning and orientation ofpolypeptides of the polypeptide of interest within the crystallizablemolecular complex, thereby facilitating crystallization of thepolypeptide of interest.

According to still further features in preferred embodiments, the atleast one molecular linker includes a region being capable offunctioning as a purification tag, the purification tag being capable offacilitating purification of the crystallizable molecular complex,and/or of facilitating the homomultimerization of the polypeptide ofinterest.

According to still further features in preferred embodiments, the regionbeing capable of functioning as a purification tag is selected from thegroup consisting of a T7 tag, a histidine tag, a Strep-tag, corestreptavidin, and biotin.

According to still further features in preferred embodiments, thepolypeptide of interest includes a region being capable of functioningas a purification tag, the purification tag being capable offacilitating purification of the crystallizable molecular complex,and/or of facilitating the homomultimerization of the polypeptide ofinterest.

According to still further features in preferred embodiments, the regionbeing capable of functioning as a purification tag is selected from thegroup consisting of a T7 tag, a histidine tag, a Strep-tag, corestreptavidin, and biotin.

According to still further features in preferred embodiments, themolecule includes a metal-binding moiety capable of specifically bindinga metal atom, the metal atom being capable of facilitatingcrystallographic analysis of the crystal.

According to still further features in preferred embodiments, themetal-binding moiety is a metal binding protein.

According to still further features in preferred embodiments, the metalbinding protein is metallothionein.

According to still further features in preferred embodiments, thepolypeptide of interest is a membrane protein.

According to still further features in preferred embodiments, themembrane protein is a G protein coupled receptor.

According to still further features in preferred embodiments, the Gprotein coupled receptor is rhodopsin or is a class A G protein coupledreceptor.

According to still further features in preferred embodiments, the classA G protein coupled receptor is m2 muscarinic cholinergic receptor.

According to still further features in preferred embodiments, thepolypeptide of interest includes a metal-binding moiety capable ofspecifically binding a metal atom, the metal atom being capable offacilitating crystallographic analysis of the crystal.

According to still further features in preferred embodiments, the metalbinding moiety is metallothionein.

According to yet another aspect of the present invention there isprovided a composition-of-matter comprising at least two molecules of amolecule-of-interest interlinked via a heterologous molecular linker,wherein the heterologous molecular linker is selected so as to definethe relative spatial positioning and orientation of the at least twomolecules within the composition-of-matter, thereby facilitatingformation of a crystal therefrom under crystallization-inducingconditions.

According to further features in preferred embodiments of the inventiondescribed below, the molecule-of-interest is a polypeptide.

According to still further features in preferred embodiments, thepolypeptide is a membrane protein.

According to still further features in preferred embodiments, themembrane protein is a G protein coupled receptor.

According to still further features in preferred embodiments, the Gprotein coupled receptor is rhodopsin or is a class A G protein coupledreceptor.

According to still further features in preferred embodiments, the classA G protein coupled receptor is m2 muscarinic cholinergic receptor.

According to still further features in preferred embodiments, theheterologous molecular linker includes at least one region capable ofspecifically binding the molecule-of-interest.

According to still further features in preferred embodiments, themolecule-of-interest is a G protein coupled receptor and the at leastone region capable of specifically binding the molecule-of-interest is amolecule selected from the group consisting of at least a portion of anarrestin molecule, at least a portion of an arrestin molecule having amutation at an amino acid residue position corresponding to position 90in bovine visual arrestin, at least a portion of an arrestin moleculehaving a mutation at an amino acid residue position corresponding toposition 175 in bovine visual arrestin, SEQ ID NO: 3, and SEQ ID NO: 4.

According to still further features in preferred embodiments, the atleast a portion of an arrestin molecule is homologous to amino acidresidues 11 to 190, or 11 to 370 of human beta-arrestin-1a.

According to still further features in preferred embodiments, the atleast a portion of an arrestin molecule comprises a G protein coupledreceptor-binding domain of the arrestin molecule.

According to still further features in preferred embodiments, themutation at an amino acid residue position corresponding to position 90in bovine visual arrestin is a mutation to a serine or threonineresidue.

According to still further features in preferred embodiments, themutation at an amino acid residue position corresponding to position 175in bovine visual arrestin is a mutation to a glutamic acid or anasparagine residue.

According to still further features in preferred embodiments, the Gprotein coupled receptor is rhodopsin or is a class A G protein coupledreceptor.

According to still further features in preferred embodiments, the classA G protein coupled receptor is m2 muscarinic cholinergic receptor.

According to still further features in preferred embodiments, theheterologous molecular linker includes a molecule selected from thegroup consisting of a polycyclic molecule, a polydentate ligand, amacrobicyclic cryptand, a polypeptide and a metal.

According to still further features in preferred embodiments, themolecule-of-interest is a G protein coupled receptor and theheterologous molecular linker comprises a molecule selected from thegroup consisting of at least a portion of an arrestin molecule, at leasta portion of an arrestin molecule having a mutation at an amino acidresidue position corresponding to position 90 in bovine visual arrestin,at least a portion of an arrestin molecule having a mutation at an aminoacid residue position corresponding to position 175 in bovine visualarrestin, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

According to still further features in preferred embodiments, the atleast a portion of an arrestin molecule is homologous to amino acidresidues 11 to 190, or 11 to 370 of human beta-arrestin-1a.

According to still further features in preferred embodiments, the atleast a portion of an arrestin molecule comprises a G protein coupledreceptor-binding domain of the arrestin molecule.

According to still further features in preferred embodiments, themutation at an amino acid residue position corresponding to position 90in bovine visual arrestin is a mutation to a serine or threonineresidue.

According to still further features in preferred embodiments, themutation at an amino acid residue position corresponding to position 175in bovine visual arrestin is a mutation to a glutamic acid or anasparagine residue.

According to still further features in preferred embodiments, the Gprotein coupled receptor is rhodopsin or is a class A G protein coupledreceptor.

According to still further features in preferred embodiments, the classA G protein coupled receptor is m2 muscarinic cholinergic receptor.

According to still further features in preferred embodiments, theheterologous molecular linker comprises core streptavidin.

According to still further features in preferred embodiments, theheterologous molecular linker includes at least two non-covalently boundsubunits.

According to still further features in preferred embodiments, theheterologous molecular linker includes a hydrophilic region, thehydrophilic region being for facilitating crystallization of themolecule-of-interest.

According to still further features in preferred embodiments, theheterologous molecular linker includes a conformationally rigid region,the conformationally rigid region being for facilitating crystallizationof the molecule-of-interest.

According to still further features in preferred embodiments, theheterologous molecular linker is selected such that thecomposition-of-matter is capable of generating a crystal selected fromthe group consisting of a 2D crystal, a helical crystal and a 3Dcrystal.

According to still further features in preferred embodiments, theheterologous molecular linker includes a metal-binding moiety capable ofspecifically binding a metal atom, the metal atom being capable offacilitating crystallographic analysis of the crystal.

According to still further features in preferred embodiments, themetal-binding moiety is a metal-binding protein.

According to still further features in preferred embodiments, the metalbinding protein is metallothionein.

According to still further features in preferred embodiments, theheterologous molecular linker includes a region being capable offunctioning as a purification tag, the purification tag being capable offacilitating purification of the crystallizable composition-of-matter,and/or of facilitating the interlinking of the at least two molecules ofa molecule-of-interest.

According to still further features in preferred embodiments, the regionbeing capable of functioning as a purification tag is selected from thegroup consisting of a T7 tag, a histidine tag, a Strep-tag, corestreptavidin, and biotin.

According to still further features in preferred embodiments, themolecule-of-interest includes a region being capable of functioning as apurification tag, the purification tag being capable of facilitatingpurification of the composition-of-matter, and/or of facilitating theinterlinking of the at least two molecules of a molecule-of-interest.

According to still further features in preferred embodiments, the regionbeing capable of functioning as a purification tag is selected from thegroup consisting of a T7 tag, a histidine tag, a Strep-tag, corestreptavidin, and biotin.

According to still further features in preferred embodiments, themolecule-of-interest includes a metal-binding moiety capable ofspecifically binding a metal atom, the metal atom being capable offacilitating crystallographic analysis of the crystal.

According to still further features in preferred embodiments, themetal-binding moiety is a metal binding protein.

According to still further features in preferred embodiments, themetal-binding protein is metallothionein.

According to still another aspect of the present invention there isprovided a nucleic acid construct comprising a polynucleotide segmentencoding a chimeric polypeptide including: (a) a first polypeptideregion being capable of specifically binding a molecule-of-interest; and(b) a second polypeptide region being capable of specifically binding ametal atom.

According to further features in preferred embodiments of the inventiondescribed below, the molecule-of-interest is a G protein coupledreceptor and the chimeric polypeptide comprises SEQ ID NO: 5 or SEQ IDNO: 6.

According to still further features in preferred embodiments, the Gprotein coupled receptor is rhodopsin or is a class A G protein coupledreceptor.

According to still further features in preferred embodiments, the classA G protein coupled receptor is m2 muscarinic cholinergic receptor.

According to still further features in preferred embodiments, themolecule-of-interest is a G protein coupled receptor and the firstpolypeptide region comprises a molecule selected from the groupconsisting of at least a portion of an arrestin molecule, at least aportion of an arrestin molecule having a mutation at an amino acidresidue position corresponding to position 90 in bovine visual arrestin,at least a portion of an arrestin molecule having a mutation at an aminoacid residue position corresponding to position 175 in bovine visualarrestin, SEQ ID NO: 3, and SEQ ID NO: 4.

According to still further features in preferred embodiments, the atleast a portion of an arrestin molecule is homologous to amino acidresidues 11 to 190, or 11 to 370 of human beta-arrestin-1a.

According to still further features in preferred embodiments, the atleast a portion of an arrestin molecule comprises a G protein coupledreceptor-binding domain of the arrestin molecule.

According to still further features in preferred embodiments, themutation at an amino acid residue position corresponding to position 90in bovine visual arrestin is a mutation to a serine or threonineresidue.

According to still further features in preferred embodiments, themutation at an amino acid residue position corresponding to position 175in bovine visual arrestin is a mutation to a glutamic acid or anasparagine residue.

According to still further features in preferred embodiments, the Gprotein coupled receptor is rhodopsin or is a class A G protein coupledreceptor.

According to still further features in preferred embodiments, the classA G protein coupled receptor is m2 muscarinic cholinergic receptor.

According to still further features in preferred embodiments, themolecule-of-interest is a polypeptide.

According to still further features in preferred embodiments, thepolypeptide is a membrane protein.

According to still further features in preferred embodiments, themembrane protein is a G protein coupled receptor.

According to still further features in preferred embodiments, the Gprotein coupled receptor is rhodopsin or is a class A G protein coupledreceptor.

According to still further features in preferred embodiments, the classA G protein coupled receptor is m2 muscarinic cholinergic receptor.

According to still further features in preferred embodiments, the secondpolypeptide region is metallothionein.

According to still further features in preferred embodiments, thechimeric polypeptide is selected such that when combined with moleculesof the molecule-of-interest under suitable conditions, the chimericpolypeptide and the molecules form a crystallizable molecular complexwhich is capable of forming a crystal containing themolecule-of-interest when subjected to crystallization-inducingconditions.

According to still further features in preferred embodiments, thechimeric polypeptide is selected such that when combined with moleculesof the molecule-of-interest and the metal atom under suitableconditions, the chimeric polypeptide and the molecules form acrystallizable molecular complex which is capable of forming a crystalcontaining the molecule-of-interest when subjected tocrystallization-inducing conditions.

According to still further features in preferred embodiments, the metalatom facilitates crystallographic analysis of the crystal.

According to still further features in preferred embodiments, thechimeric polypeptide includes a hydrophilic region, the hydrophilicregion being for facilitating crystallization of themolecule-of-interest.

According to still further features in preferred embodiments, thechimeric polypeptide includes a conformationally rigid region, theconformationally rigid region being for facilitating crystallization ofthe molecule-of-interest.

According to still further features in preferred embodiments, thechimeric polypeptide is selected so as to define the spatial positioningand orientation of the molecule-of-interest within the crystallizablemolecular complex, thereby facilitating crystallization of themolecule-of-interest.

According to still further features in preferred embodiments, thechimeric polypeptide is selected such that the crystallizable molecularcomplex formed is capable of generating a crystal selected from thegroup consisting of a 2D crystal, a helical crystal and a 3D crystal.

According to still further features in preferred embodiments, thechimeric polypeptide further includes a polypeptide region being capableof functioning as a purification tag, the purification tag being capableof facilitating purification of the crystallizable molecular complex,and/or of facilitating the binding of a molecule-of-interest.

According to still further features in preferred embodiments, the regionbeing capable of functioning as a purification tag is selected from thegroup consisting of a T7 tag, a histidine tag, a Strep-tag, corestreptavidin, and biotin.

According to a further aspect of the present invention there is provideda nucleic acid construct comprising a polynucleotide segment encoding achimeric polypeptide including: (a) a first polypeptide region beingcapable of specifically binding a molecule-of-interest; (b) a secondpolypeptide region being capable of homomultimerization into a complexof defined geometry; and (c) a third polypeptide region being capable ofspecifically binding a metal atom.

According to further features in preferred embodiments of the inventiondescribed below, the molecule-of-interest is a G protein coupledreceptor and the first polypeptide region is selected from the groupconsisting of at least a portion of an arrestin molecule, at least aportion of an arrestin molecule having a mutation at an amino acidresidue position corresponding to position 90 in bovine visual arrestin,at least a portion of an arrestin molecule having a mutation at an aminoacid residue position corresponding to position 175 in bovine visualarrestin, SEQ ID NO: 3, and SEQ ID NO: 4.

According to still further features in preferred embodiments, the atleast a portion of an arrestin molecule is homologous to amino acidresidues 11 to 190, or 11 to 370 of human beta-arrestin-1a.

According to still further features in preferred embodiments, the atleast a portion of an arrestin molecule comprises a G protein coupledreceptor-binding domain of the arrestin molecule.

According to still further features in preferred embodiments, themutation at an amino acid residue position corresponding to position 90in bovine visual arrestin is a mutation to a serine or threonineresidue.

According to still further features in preferred embodiments, themutation at an amino acid residue position corresponding to position 175in bovine visual arrestin is a mutation to a glutamic acid or anasparagine residue.

According to still further features in preferred embodiments, the Gprotein coupled receptor is rhodopsin or is a class A G protein coupledreceptor.

According to still further features in preferred embodiments, the classA G protein coupled receptor is m2 muscarinic cholinergic receptor.

According to still further features in preferred embodiments, the secondpolypeptide region comprises core streptavidin.

According to still further features in preferred embodiments, themolecule-of-interest is a G protein coupled receptor and the chimericpolypeptide comprises SEQ ID NO: 5 or SEQ ID NO: 6.

According to still further features in preferred embodiments, the Gprotein coupled receptor is rhodopsin or is a class A G protein coupledreceptor.

According to still further features in preferred embodiments, the classA G protein coupled receptor is m2 muscarinic cholinergic receptor.

According to still further features in preferred embodiments, the thirdpolypeptide region comprises metallothionein.

According to still further features in preferred embodiments, themolecule-of-interest is a polypeptide.

According to still further features in preferred embodiments, thepolypeptide is a membrane protein.

According to still further features in preferred embodiments, themembrane protein is a G protein coupled receptor.

According to still further features in preferred embodiments, the Gprotein coupled receptor is rhodopsin or is a class A G protein coupledreceptor.

According to still further features in preferred embodiments, the classA G protein coupled receptor is m2 muscarinic cholinergic receptor.

According to still further features in preferred embodiments, thechimeric polypeptide is selected such that when combined with moleculesof the molecule-of-interest, the chimeric polypeptide and the moleculesform a crystallizable molecular complex of defined geometry which iscapable of forming a crystal containing the molecule-of-interest whensubjected to crystallization-inducing conditions.

According to still further features in preferred embodiments, thechimeric polypeptide includes a hydrophilic region, the hydrophilicregion being for facilitating crystallization of themolecule-of-interest.

According to still further features in preferred embodiments, thechimeric polypeptide includes a conformationally rigid region, theconformationally rigid region being for facilitating crystallization ofthe molecule-of-interest.

According to still further features in preferred embodiments, thechimeric polypeptide is selected so as to define the spatial positioningand orientation of molecules of the molecule-of-interest within thecrystallizable molecular complex, thereby facilitating crystallizationof the molecule-of-interest.

According to still further features in preferred embodiments, thechimeric polypeptide is selected such that the crystallizable molecularcomplex of defined geometry formed is capable of generating a crystalselected from the group consisting of a 2D crystal, a helical crystaland a 3D crystal.

According to still further features in preferred embodiments, the metalatom facilitates crystallographic analysis of the molecule-of-interestcontained in the crystal.

According to still further features in preferred embodiments, thechimeric polypeptide further includes a polypeptide region being capableof functioning as a purification tag, the purification tag being capableof facilitating purification of the crystallizable molecular complex,and/or of facilitating the binding of a molecule-of-interest.

According to still further features in preferred embodiments, the regionbeing capable of functioning as a purification tag is selected from thegroup consisting of a T7 tag, a histidine tag, a Strep-tag, and corestreptavidin.

According to a yet a further aspect of the present invention there isprovided a method of purifying a G protein coupled receptor from asample containing the G protein coupled receptor, the method comprisingsubjecting the sample to affinity chromatography using an affinityligand selected from the group consisting of at least a portion of anarrestin molecule, at least a portion of an arrestin molecule having amutation at an amino acid residue position corresponding to position 90in bovine visual arrestin, at least a portion of an arrestin moleculehaving a mutation at an amino acid residue position corresponding toposition 175 in bovine visual arrestin, a molecule defined by SEQ ID NO:3, and a molecule defined by SEQ ID NO: 4, thereby purifying the Gprotein coupled receptor.

According to further features in preferred embodiments of the inventiondescribed below, the at least a portion of an arrestin molecule ishomologous to amino acid residues 11 to 190, or 11 to 370 of humanbeta-arrestin-1a.

According to still further features in preferred embodiments, the atleast a portion of an arrestin molecule comprises a G protein coupledreceptor-binding domain of the arrestin molecule.

According to still further features in preferred embodiments, themutation at an amino acid residue position corresponding to position 90in bovine visual arrestin is a mutation to a serine or threonineresidue.

According to still further features in preferred embodiments, themutation at an amino acid residue position corresponding to position 175in bovine visual arrestin is a mutation to a glutamic acid or anasparagine residue.

According to still further features in preferred embodiments, the Gprotein coupled receptor is rhodopsin or is a class A G protein coupledreceptor.

According to still further features in preferred embodiments, the classA G protein coupled receptor is m2 muscarinic cholinergic receptor.

According to still further features in preferred embodiments, theaffinity ligand includes a region being capable of functioning as apurification tag, the purification tag being capable of facilitatingattachment of the affinity ligand to an affinity chromatography matrix.

According to still further features in preferred embodiments, the regionbeing capable of functioning as a purification tag is selected from thegroup consisting of a T7 tag, a histidine tag, a Strep-tag, corestreptavidin, and biotin.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only and are presented inthe cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 a is a diagram depicting the general configuration of anon-polypeptidic molecular linker which can be used for multimerizationof a molecule-of-interest according to the teachings of the presentinvention. MS: molecular scaffold, M: metal atom; L: linking chaincontaining 1-3 carbon or oxygen atoms (shown in FIG. 1 b); G=[—CO₂],[—CONH], [—O], [—OCO] or [—NHCO]; L′=linking chain of 1-10 atomscontaining carbon or oxygen atoms, such as [(CH₂CH₂O)₂—O—CH₂CH₂—] or[—(CH₂)₄—]; SBD=specific binding domain, such as [—N⁺(CH₃)₃] or[—CO(CF₃)], or a polypeptide such as biotin.

FIG. 1 b is a diagram depicting a linking chain containing 1-3 carbon oroxygen atoms comprised in the non-polypeptidic molecular linkerdescribed in FIG. 1 a. G′=[CO₂H], [OH] or [NH₂].

FIGS. 2 a-b are diagrams depicting porphyrin-based molecular linkerswhich can be used according to the teachings of the present inventionfor multimerization of two (FIG. 2 a) or four (FIG. 2 b) molecules ofinterest. X=[L-G-L′-SBD], as defined in FIG. 1 a; R=H, (sub)-phenyl or[L-G-L′-SBD], as defined in FIG. 1 a, M=metal atom.

FIG. 3 is a diagram depicting a hydroxime-based molecular linker whichcan be used according to the teachings of the present invention formultimerization of two molecules of interest. X=[L-G-L′-SBD], R′=H,(sub)-phenyl or [L-G-L′-SBD], as defined in FIG. 1 a; R′=H or methylgroup; M=metal atom.

FIGS. 4 a-b are schematic diagrams depicting synthesis of the porphyrinmolecular linkers of FIGS. 2 a-b which can be used for multimerizationof four (FIG. 4 a) or two (FIG. 4 b) molecules of interest. HY=a strongacid; MZ₂=a transition or heavy metal salt; Oxid=an oxidant, such as DDQor O₂.

FIG. 5 is a schematic diagram depicting synthesis of the hydroxime-basedmolecular linker of FIG. 3. MZ₂=a transition or heavy metal salt.

FIG. 6 a is a schematic diagram depicting linkage of a biotinylatedmoiety to porphyrin-based molecular linkers such as those depicted inFIGS. 2 a-b.

FIG. 6 b is a schematic diagram depicting linkage of a trimethylammoniummoiety to hydroxime-based molecular linkers such as the one depicted inFIG. 3. MZ₂=a transition or heavy metal salt.

FIGS. 7 a-b are schematic diagrams depicting polynucleotide constructsfor purification of molecules of interest. FIG. 7 a is a diagramdepicting a construct encoding a chimeric polypeptide containing asingle-chain Fv (scFv) segment fused to a core streptavidin andpurification tag segments. FIG. 7 b is a diagram depicting a constructencoding a chimeric polypeptide containing a Strep-tag (Stag) segmentfused to a metal atom binding polypeptide (MBP) segment fused in turn toa purification tag segment. The relative positions of the Strep-tag andmetal atom binding polypeptide can also be inverted. NH₂-amino-terminus;leader-leader sequence or signal peptide for expression in eukaryotic orprokaryotic cells; V_(H) and V_(L)-antibody variable heavy and lightchains, respectively.

FIG. 8 is a diagram depicting the conformation of a core-streptavidintetramer used in the molecular linkers of the present inventionindicating the N-terminal fusion sites thereof for attachment ofmoieties capable of specifically binding a molecule-of-interest, such asa single-chain Fv, and the binding site for attachment of a Strep-tag ora biotin moiety.

FIGS. 9 a-b are sequence diagrams depicting the amino acid residuesequence of portions of human beta-arrestin-1a suitable for bindingdifferent classes of GPCRs with high affinity and specificityindependently of the phosphorylation-activation state thereof. FIG. 9 adepicts a polypeptide composed of amino acid residues 11-190 of humanbeta-arrestin-1a with mutation R169E. FIG. 9 b depicts a polypeptidecomposed of amino acid residues 11-370 of human beta-arrestin-1a withmutation R169E. In both polypeptides, mutation R169E conferring thecapacity to bind GPCRs independently of the phosphorylation-activationstate thereof, and the wild type serine residue at position 86conferring the capacity to bind multiple types of GPCRs are indicated(bold underlined).

FIGS. 10 a-b are sequence diagrams depicting the amino acid residuesequence of molecular linkers for crystallization of different classesof GPCRs independently of the phosphorylation-activation state thereof.FIG. 10 a depicts a linker composed of a chimeric protein consisting ofthe N- to C-terminal segments; T7 tag (N-terminal italics), corestreptavidin (uppercase), the peptide linker GSAA (SEQ ID NO: 1;internal italics), and amino acid residues 11-190 of humanbeta-arrestin-1a (lowercase) with mutation R169E. FIG. 10 b depicts alinker composed of a chimeric protein consisting of the N- to C-terminalsegments; T7 tag (N-terminal italics), core streptavidin (uppercase),the peptide linker GSAA (SEQ ID NO: 1; internal italics), and amino acidresidues 11-370 of human beta-arrestin-1a (lowercase) with mutationR169E. In the arrestin derived segment of both molecular linkers,mutation R169E conferring the capacity to bind GPCRs independently ofthe phosphorylation-activation state thereof, and the wild type serineresidue at position 86 conferring the capacity to bind multiple types ofGPCRs are indicated (bold underlined).

FIG. 11 is a chemical structure diagram depicting a porphyrin-NTA-Ni²⁺molecular linker used for crystallization of histidine-tagged proteins.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of methods and compositions which can be usedfor generating crystals containing a molecule-of-interest, and ofmethods of purifying G protein coupled receptors (GPCRs). Specifically,the present invention can be used to generate crystals of membraneproteins which can be used to determine the three-dimensional (3D)atomic structure thereof, and to purify GPCRs using arrestin derivedpolypeptides as affinity ligands of GPCRs.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description or illustrated in the drawings. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

Various methods of assisting the crystallization of molecules such aspolypeptides and of facilitating their crystallographic analysis havebeen described in the prior art.

Techniques involving protein modifications, such as those based onfusion of the polypeptide of interest to a large heterologoushydrophobic polypeptide domain, alteration and engineering of crystalunit cell contacts or complexation of a protein of interest withantibody fragments are typically dedicated, labor intensive and requiremuch fine tuning. In addition, methods relying on artificialfunctionalized lipid scaffolds are only useful for the creation ofplanar 2D crystals which can be studied by electron microscopy, but notby X-ray diffraction, or are useful for generation of helical crystalswhich do not permit high resolution 3D structural analysis.

Thus, prior art approaches for assisting or facilitating crystallizationof molecules-of-interest have failed to provide adequate solutions forthe controlled 3D crystallization of molecules such as polypeptides,while allowing subsequent determination of their 3D atomic structure.

In sharp contrast to prior art techniques, the methods of the presentinvention enable the generation of readily crystallizable molecularcomplexes incorporating molecules of a molecule-of-interest, such as amembrane protein. In addition, the present invention also enablespurification of the molecule-of-interest, thereby greatly facilitatingcrystallographic analysis thereof.

Thus, according to the present invention, there is provided a method ofgenerating a 2D, or preferably a 3D, crystal containing amolecule-of-interest.

According to one embodiment of the method of the present invention,crystallization of a molecule-of-interest is effected by contactingmolecules of the molecule-of-interest with at least one type of linker.The linker is selected so as to be capable of interlinking at least twomolecules of the molecule-of-interest to thereby form a crystallizablemolecular complex of defined geometry (defined spatial orientation). Asis further described hereinunder, the linker can be composed of a singlemolecule or a complex including a plurality of molecules, depending onthe application and purpose.

Following linker-molecule-of-interest binding, the molecular complexformed is subjected to crystallization-inducing conditions, such asthose described in Example 6 of the Examples section, thereby generatingthe crystal containing the molecule-of-interest.

As mentioned hereinabove, both single molecule and multi-molecule linkerconfigurations can be used by the present invention.

A single-molecule linker can include binding regions covalently attachedto a core, while a multi-molecule linker (linker complex) can includebinding regions non-covalently associated with a core unit, and/or mayinclude a core unit composed of non-covalently associated subunits. Inany case, the linker is designed and configured such that when complexedwith molecules of a molecule-of-interest, the linker directs the spatialorientation of the molecules of the molecule-of-interest so as to form amolecular complex of pre-defined geometry, thereby facilitatingcrystallization of the molecule-of-interest when the molecular complexis subjected to crystallization inducing conditions. The followingExamples section describes specific examples of single-molecule andmulti-molecule type linkers, as further detailed hereinbelow.

As used herein, a “core” of a linker refers to a portion of the linkerfunctioning as the basic molecule-of-interest multimerization scaffoldof the linker.

Regardless of core configuration, minimizing core size may beadvantageous depending on the application and purpose. Cores of minimalsize may be generally advantageous since this may minimize the size ofthe linker, which in turn serves to maximize tightness of packing of themolecular complex. This minimizes conformational disorder in themolecular complex, thus ensuring optimal ordering of crystals. As afurther advantage, minimizing core size may make the linker easierand/or cheaper to produce and purify.

Single molecule linkers, being composed of covalently connected atoms,are highly stable and rigid and can be advantageously used to generatemolecular complexes having minimized conformational disorder, forexample, relative to linker complexes. Thus, single molecule linkers canbe used to generate optimally ordered crystals, and may be moreconveniently, cheaply, and/or easily produced relative to linkercomplexes.

Linker complexes may advantageously comprise homomultimerized proteins,such as, for example, fusion proteins comprising a homomultimerizingdomain and a polypeptide or polypeptides, such as a binding domainand/or a purification tag, being capable of facilitating crystallizationand/or 3D structure determination of a molecular complex, as furtherdescribed hereinbelow. The use of linker complexes comprising suchhomomultimerized fusion proteins may be advantageously employed toobviate the need to separately express the polypeptide components ofsuch fusion proteins, as well as the need to subject such components toconditions facilitating their association, thereby greatly facilitatinggeneration of the linker complex, generation of the molecular complex,and/or crystallization of a molecule-of-interest.

The linkers of the present invention include one or preferably severalbinding domains for specifically binding the molecule-of-interest. Suchbinding domains can be synthesized as part of the linker or as distinctmolecules which can be non-covalently associated with a core molecule toform the linker (linker complex).

Non-covalent association of binding domains to linkers can beadvantageously used to enable the linkers of the present invention to bemodular, such that one type of molecular linker core can be used toassociate essentially any desired binding domain according to the targetmolecule to be complexed and crystallized.

Binding domains which bind molecules of a molecule-of-interestcovalently or binding domains which bind molecules of amolecule-of-interest non-covalently can be used, depending on theapplication and purpose.

Binding domains which bind a molecule-of-interest non-covalently can beadvantageously used to bind a molecule-of-interest without the need toresort to chemical synthesis techniques required for covalently couplingmolecules. In the case of a biomolecular molecule-of-interest, theavailability of highly specific ligands, such as, for example,antibodies, provides a pool of molecules useable as highly efficientbinding domains.

Binding domains which bind a molecule-of-interest covalently can beadvantageously used to bind a molecule-of-interest with great stability,thereby minimizing conformational disorder in crystals generatedtherewith, relative, for example, to binding domains which bind amolecule-of-interest non-covalently.

Preferably, single molecule linkers are porphyrin based. Porphyrin basedlinkers can be advantageously used to multimerize molecules of amolecule-of-interest with great stability and rigidity, as described inExample 1 of the following Examples section.

Multimerized streptavidin or streptavidin derived molecules may beadvantageously utilized as the core of a molecular linker.

Preferably, the streptavidin molecule or streptavidin derived moleculeis a core streptavidin. Suitable core streptavidins may comprise, forexample, amino acid residues 13-133, 13-131 or 16-131 of nativestreptavidin.

The use of core streptavidin as the core of molecular linkers isadvantageous since core streptavidin homomultimerizes into aparticularly tightly packed tetramer, for example relative to nativestreptavidin tetramer. As a further advantage, core streptavidintetramers display enhanced stability under denaturing conditions, andtheir biotin binding sites appear to be more accessible relative tonative streptavidin tetramer. Extensive literature exists for theexpression, purification and uses of streptavidin or streptavidinderived molecules (Wu S C. et al., 2002. Protein Expression andPurification 24:348-356; Gallizia A. et al., 1998. Protein Expressionand Purification 14:192-196), fusion proteins comprising streptavidin orstreptavidin derived molecules (Sano T. & Cantor C R. 2000. MethodsEnzymol. 326:305-11), and modified streptavidin or streptavidin derivedmolecules (see, for example: Sano T. et al., 1993. Journal of BiologicalChemistry 270:28204-28209), including for streptavidin or streptavidinderived molecules whose gene sequence has been optimized for expressionin E. coli (Thompson L D. & Weber P C., 1993. Gene 136:243-6).

Fusion proteins comprising core streptavidins may be optimal whencomprising an N-terminal core streptavidin segment and/or when producedas inclusion bodies. This may optimize correct folding and/or maximizethe number of free biotin binding sites.

Molecular linkers including multimerized fusion proteins comprising corestreptavidin and a polypeptidic binding domain, such as a single chainantibody Fv or a biological ligand of the molecule-of-interest, can beconveniently used to efficiently crystallize a molecule-of-interest.

Synthesis of chimeric polypeptides comprising core streptavidin and asingle chain Fv can be effected by cloning nucleic acid sequencesencoding the single chain Fv in an expression vector configured toexpress an in-frame chimeric polypeptide comprising core streptavidin,and the single chain Fv in a suitable host such as E. coli followingtransformation thereof using standard recombinant polypeptide expressiontechnology.

Detailed protocols for the synthesis of streptavidin-single chain Fvfusion proteins can be found in the literature of the art (for examplerefer to Cloutier S M. et al., 2000. Molecular Immunology 37:1067-1077;Dubel S. et al., 1995. J Immunol Methods 178:201; Huston J S. et al.,1991. Methods in Enzymology 203:46; Kipriyanov S M. et al., 1995. HumAntibodies Hybridomas 6:93; Kipriyanov S M. et al., 1996. ProteinEngineering 9:203; Pearce L A. et al., 1997. Biochem Molec Biol Intl42:1179-1188).

As is shown in Examples 7 and 9 of the Examples section which follows,core streptavidin based molecular linkers can be used to crystallize amolecule-of-interest.

Suitable binding domains which bind a molecule-of-interestnon-covalently include but are not limited to, polypeptides derived fromantibodies, such as, for example, single-chain Fv fragments, asdescribed in Example 7 of the Examples section, T cell receptors,MHC-peptide complexes, biological ligands of the molecule-of-interest,and affinity-selected peptides, such as phage-display selected peptides.

As described in Example 7 of the Examples section, single-chain Fvfragments can be advantageously used to specifically bind andcrystallize a molecule-of-interest.

In general, synthesis a single chain Fv molecule specific for amolecule-of-interest comprises producing and screening hybridoma celllines secreting an antibody specific for the molecule-of-interest viastandard hybridoma production techniques, and using RT-PCR to clone cDNAsequences encoding the variable light and variable heavy chains of theantibody. Ample guidance regarding production of single chain Fv's andfusion proteins comprising single chain Fv's is available in theliterature of the art.

Suitable binding domains which bind a molecule-of-interest covalentlyinclude various chemical groups such as, for example, [—N⁺(CH₃)₃] and[—CO(CF₃)] (trifluorocarbonyl), as described in Example 1 of theExamples section, and N-(5-amino-1-carboxypentyl)imino-diacetic acid(NTA), as described in Example 11 of the following Examples section.Covalent coupling of a molecule-of-interest to a linker can be effectedusing standard chemical techniques for which guidance is broadlyavailable in the literature of the art. For example, a trifluorocarbonylgroup can be bound to the amino end, as well as to amino acid residueshaving free —OH, —SH, —NH2 groups of a polypeptidicmolecule-of-interest, via a reaction of these groups with a compoundsuch as HO—C(═O)—CF₃, under appropriate conditions.

It will be appreciated that other than as described hereinabove, linkeruniversality can also be achieved by modifying the molecule to becrystallized to include specific binding moieties recognized by a singleand universal linker, for example as described in Example 8 of theExamples section below. In the case of a polypeptidicmolecule-of-interest, the molecule-of-interest can be expressed as partof a chimeric polypeptide including the binding moiety. Alternatively,the moiety is chemically attached to the molecule-of-interest. In anycase, the binding moiety is preferably selected such that it readilyassociates with the linker while not substantially modifying thestructure of the molecule to be crystallized.

Examples of binding domains of such universal linkers include biotin, asdescribed in Examples 2 and 4 of the Examples section, anantibody-derived molecule, such as an anti purification tag single-chainFv fragment, as described in Example 7 of the Examples section, a nickelion, as described in Example 11 of the Examples section below, oressentially any specific ligand of a purification tag.

Examples of moieties which can be used to modify a molecule-of-interestsuch that it may be bound by universal linkers comprising specificligands of purification tags include various purification tags.

As used herein, the term “purification tags” encompasses affinity tags.

Examples of purification tags include epitope tags, histidine tags,Strep-tags, single-chain Fv molecules, core streptavidin, streptavidin,and biotin.

Guidance regarding tagging molecules with histidine tags, and uses ofsuch molecules is available in the literature of the art (for example,refer to: Sheibani N. 1999. Prep Biochem Biotechnol. 29:77).

Guidance regarding tagging molecules with Strep-tags, and uses of suchmolecules is available in the literature of the art (for example, referto: Schmidt, T G M. and Skerra, A. Protein Eng. 1993, 6:109; Skerra A. &Schmidt T G M., 1999. Biomolecular Engineering 16:79-86).

Epitope tags can be comprised in a molecule-of-interest to enablecomplexation with linkers comprising single-chain Fv domains specificfor such epitope tags.

Examples of epitope tags include an 11-mer Herpes simplex virusglycoprotein D peptide, and an 11-mer N-terminal bacteriophage t7peptide, being commercially known as HSVTag and T7 Tag, respectively(Novagen, Madison, Wis., USA), and 10- or 9-amino acid c-myc orHemophilus influenza hemagglutinin (HA) peptides, which are recognizedby the variable regions of monoclonal antibodies 9E10 and 12Ca5,respectively.

Examples of moieties which can be used to modify molecules of interestsuch that these may be bound by a linker comprising biotin includestreptavidin, core streptavidin and anti biotin single-chain antibodyFv.

Examples of moieties which can be used to modify molecules of interestsuch that these may be bound by a linker comprising streptavidin includeStrep-tags, as described in Example 8 of the Examples section, orbiotin.

Examples of moieties which can be used to modify molecules of interestsuch that these may be bound by a linker comprising a metal atominclude, but are not limited to, histidine tags.

In the case of polypeptidic molecules-of-interest, polypeptide tags,such as, for example, histidine tags or Strep-tags, are particularlyconvenient since the molecule-of-interest and the tag can beco-expressed as a chimeric protein.

As mentioned hereinabove, the linkers of the present inventionfacilitate crystallization of molecules of interest by enabling thegeneration of a molecule-linker complex in which bound molecules arepositioned in a defined spatial orientation. To allow such spatialpositioning, the linker is selected of a size and geometricconfiguration which is capable of restricting the bound molecules to apredetermined orientation thus greatly facilitating 3D crystalformation.

Linker size and geometric configuration selection are also influenced bythe need to maximize molecule-molecule interactions during or followingcomplex formation. Such molecule-molecule interactions enhance thestability of the complex formed and thus further facilitate crystalformation therefrom.

It will be appreciated that linker length and spatial configurationselection is effected in accordance with the molecule to becrystallized. Such selection may be advantageously facilitated usingcomputerized 3D modeling of the assembled crystallization complex. Suchcomputerized 3D modeling is routinely effected by the ordinarily skilledpractitioner using software available via the Internet/World Wide Web.Suitable software applications which may be used to generate 3Dstructure models of molecules include RIBBONS (Carson, M. (1997) Methodsin Enzymology 277: 25), O (Jones, T A. et al. (1991) Acta CrystallogrA47:110), DINO (DINO: Visualizing Structural Biology (2001)http://www.dino3d.org); and QUANTA, CHARMM, INSIGHT, SYBYL, MACROMODE,ICM, MOLMOL, RASMOL and GRASP (reviewed in Kraulis, J. (1991) ApplCrystallogr. 24:946).

For example, in the case of membrane proteins, a corestreptavidin-single-chain Fv linker (Example 7) can be used totetramerize a membrane protein to form a non-planar geometricconfiguration. Such a non-planar geometric configuration would preventthe membrane protein from forming disordered aggregates or 2D crystalsand would thus enable the generation of 3t) crystals therefrom.

In the case of molecules which lack sufficient conformational rigidity,the linkers employed are designed so as to provide rigidity to boundmolecules thereby further facilitating crystallization thereof.

Such conformational rigidity can be obtained by utilizing linkers havingcores based on polydentate ligands, including, but not limited to,polydentate ligands, such as porphyrin, or macrobicyclic cryptands, suchas hydroxime, as described in Examples 1-5 and 11 of the Examplessection which follows. As described hereinabove, core streptavidintetramer can be used to generate a suitably conformationally rigidlinker.

In addition to the above described features, the linkers employed by thepresent invention can also include several additional features.

According to another preferred embodiment of the present invention, thelinkers include a hydrophilic domain such that complexes formed therebyare sufficiently hydrophilic so as to facilitate crystallization ofmolecules of interest which are substantially hydrophobic.

Examples of such “hydrophilic” linkers include, for example, linkerscomprising core streptavidin or single-chain Fv, as described in Example7 of the Examples section, linkers comprising non-polypeptidichydrophilic molecules such as, for example, trimethylammonium, asdescribed in Example 5 of the Examples section, or linkers comprisingN-(5-amino-1-carboxypentyl)imino-diacetic acid (NTA) groups, asdescribed in Example 11 of the Examples section below.

According to another preferred embodiment of the present invention, thelinkers include a purification tag, for example, as describedhereinabove. Such a purification tag can be advantageously used forpurification of the linker and/or of the molecular complex.

Purification of a molecule-of-interest is a critical and limiting stepin the crystallization of a molecule-of-interest, such as a polypeptidicmolecule-of-interest and, as such, methods for improving suchpurification can serve to thereby greatly facilitate the crystallizationof such molecules of interest. The same considerations may be applicableto purification of the linkers, such as the polypeptide-based linkers ofthe present invention.

Examples of suitable purification tags include, for example, the epitopetags to which specific antibodies exist which are listed and describedhereinabove, a Strep-tag and a histidine tag, as described in Example 7of the Examples section. Purification of a molecule containing ahistidine tag is routinely performed by those well-versed in the art,using nickel-based automatic affinity column purification techniques.Purification of a molecule containing a Strep-tag can be effected usingstandardized techniques, for example, as described hereinabove.

The method of the present invention can be used to crystallize any knowntype of molecules including inorganic and organic molecules.

Examples of organic molecules include, but are not limited to,polypeptides such as membrane proteins, receptors, enzymes, antibodiesand prions, as well as nucleic acids, carbohydrates, hormones,polycyclic molecules and lipids.

The present invention can be advantageously used to crystallize a GPCR.

Preferably, the present invention is used to crystallize a GPCR such asrhodopsin or a class A GPCR.

Preferably, the present invention is used to crystallize a class A GPCRsuch as m2 muscarinic cholinergic receptor.

Guidance regarding families, types or classes of GPCRs, including mutantGPCRs, is widely available in the literature of the art (see, forexample: Edvardsen O. et al., 2002. Nucleic Acids Res. 30:361; Attwood TK. et al., 2002. Protein Eng. 15(1):7)

Crystallization of GPCRs is preferably effected using molecular linkerscomprising as a binding domain a GPCR-binding domain of an arrestinmolecule.

Types of arrestins which can be used according to the method of thepresent invention include, but are not limited to, beta-arrestin-1a(Lohse M J. et al., 1990. Science 248:1547-1550; Parruti, G. et al.,1993. J Biol Chem. 268:9753-9761; Calabrese G. et al., 1994. Genomics24:169-171; Lefkowitz R J., 1998. J Biol Chem. 273:18677-18680; LuttrellL M. et al., 1999. Science 283:655-661), arrestin-C (Craft C M. et al.,1994. J Biol Chem. 269:4613-4619), S-arrestin (Yamaki K. et al., 1990. JBiol Chem. 265:20757-20762; Calabrese G. et al., 1994. Genomics23:286-288; Yamamoto S. et al., 1997. Nat Genet. 15:175-178; Sippel K C.et al., 1998. Invest Ophthalmol Vis Sci. 39:665-670), arrestin 3(Murakami A. et al., 1993. FEBS Lett. 334:203-209; Craft C M. et al.,1994. J Biol Chem. 269:4613-4619; Sakuma H. et al., 1996. FEBS Lett.382:105-110), beta-arrestin-2 (Rapoport B. et al., 1992. Mol CellEndocrinol. 84:R39-R43; Attramadal H. et al., 1992. J Biol Chem.267:17882-17890; Calabrese G. et al., 1994. Genomics 23:286-288;Lefkowitz R J., 1998. J Biol Chem. 273:18677-18680), andbeta-arrestin-1b (Lohse M J. et al., 1990. Science 248:1547-1550;Parruti G. et al., 1993. J Biol Chem. 268:9753-9761; Calabrese G. etal., 1994. Genomics 24:169-171; Lefkowitz R J., 1998. J Biol Chem.273:18677-18680; Luttrell L M. et al., 1999. Science 283:655-661). Ampleguidance regarding the location of G protein coupled receptor bindingdomains of arrestins is provided in the aforementioned references and inthe Examples section which follows.

Preferably, the arrestin molecule is beta-arrestin-1a.

Regardless of the type of arrestin used, the GPCR binding domain ispreferably homologous to amino acid residues 11 to 190, or 11 to 370 ofhuman beta-arrestin-1a.

Preferably, the G protein coupled receptor-binding domain has a mutationat an amino acid residue position corresponding to position 90 in bovinevisual arrestin, a mutation at an amino acid residue positioncorresponding to position 175 in bovine visual arrestin, or morepreferably both.

Preferably, the mutation at an amino acid residue position correspondingto position 90 in bovine visual arrestin is a mutation to a threonineresidue or more preferably to a serine residue.

Preferably, the mutation at an amino acid residue position correspondingto position 175 in bovine visual arrestin is a mutation to a anasparagine residue or more preferably to a glutamic acid residue.

Guidance regarding identification of amino acid residue positions invarious arrestins corresponding to amino acid residue positions inbovine visual arrestin can be found in the literature of the art (see,for example: Han M. et al., 2001. Structure (Camb) 9:869-80; Hirsch J A.et al., 1999. Cell 97:257-69).

In general, corresponding amino acid residue positions between any pairof related proteins, such as a pair of arresting, may be computationallydetermined using software tools suitable for aligning proteins, such asalignment software of the NCBI available on the World Wide Web/Internet.

As is described in Example 9 of the following Examples section,GPCR-binding domains of arrestins having a serine residue at an aminoacid residue position corresponding to position 90, or a glutamic acidresidue an amino acid residue position corresponding to position 175 inbovine visual arrestin can, respectively, be advantageously used to binddifferent types of GPCRs or to bind GPCR independently of itsactivation-phosphorylation state, respectively.

Preferably, the GPCR binding domain corresponds to the amino acidsequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4. As shown in Example9 of the Examples section below molecular linkers comprising SEQ ID NO:3 or SEQ ID NO: 4 can be used to specifically bind various types ofGPCRs with high affinity and specificity regardless of the activationstate of such GPCRs.

Crystallization of the linker-molecule complex can be effected via anyof the standard means described in the literature, including, forexample, microbatch, vapor diffusion or dialysis (Bergfors, T. M.,Protein crystallization. IUL Biotechnology Series. 1999, La Jolla,Calif.: International University Line). In such methods, the appropriateamount of linker is added to a monodisperse solution of themolecule-of-interest and the solution is then employed in any of themethods mentioned above. For example, the optimal amount of reagents,such as linker subunits, to be added for facilitating crystallizationcan be determined by dynamic light scattering so as to ensuremonodispersity of the crystallizable molecular complex and to measurethe second virial coefficient, which can be employed as a diagnosticindicator for the tendency of the molecular species in solution tocrystallize (George, A., et al., Macromolecular Crystallography, Pt a.1997. p. 100).

To facilitate X-ray crystallographic determination of the structure of acrystallized molecule-of-interest, the molecular complexes of thepresent invention can further include at least one metal atom associatedtherewith. Such a metal atom can be used to generate initial phases forX-ray diffraction crystallography, via methodologies such as multipleanomalous diffraction (MAD) (Hendrickson W A., Science 1991, 254:51),thereby facilitating solution, for example, of the 3D atomic structureof the crystallized molecule.

Alternately, X-ray crystallographic structure determination of themolecule-of-interest may be facilitated by association of a metal atomwith the molecule-of-interest.

Examples of such metal atoms include, for example, iron, cobalt, nickel,cadmium, platinum and zinc.

To be capable of associating with a metal atom, the linkers of thepresent invention may include polydentate ligands, such as porphyrin,and macrobicyclic cryptands, such as hydroxime, as described in Example1 of the Examples section.

Alternately, to be capable of associating with a metal atom, the linkersof the present invention or a molecule-of-interest may include, forexample, a metal binding protein, such as metallothionein,desulforedoxin, rubredoxin, colicin or rubrerythrin.

Preferably, the metal binding protein is metallothionein.

Conjugation of a metal binding protein with a polypeptidic linker ormolecule-of-interest can be conveniently effected by co-expressing themetal binding protein with the linker or the molecule-of-interest as afusion protein.

For example, metallothionein-streptavidin fusion proteins may begenerated as previously described (Sano T. et al., 1999. Proc Natl AcadSci USA. 89:1534-8).

As shown in Example 9 of the Examples section below, a molecular linkercomprising metallothionein can be used to generate a highly orderedcrystal of a membrane protein, which crystal comprising a metal atomuseful for determining initial phases for structural analysis of such amembrane protein.

It will be understood by one versed in the art that metal atomsfacilitating crystallographic analysis, as described in the presentinvention, include the ionized forms of such metal atoms, such as, forexample, Pt²⁺, Ni²⁺, Cu²⁺ or Co²⁺.

It will be appreciated that such a metal atom can also serve as anucleating core around which linker arms can associate into a linkercomplex as described hereinabove.

Thus, the present invention enables crystallization of anymolecule-of-interest and, in particular, hydrophobic and amphiphilicmolecules which are difficult or impossible to crystallize using priorart methods.

In sharp contrast to the linkers used by prior art methods, the linkerconfigurations used by the method of the present invention:

(i) are capable of forming molecular complexes with molecules ofinterest of a sufficient solubility so as to facilitate crystallizationthereof,

(ii) can be easily modified to include binding moieties specific forvirtually any region of any molecule-of-interest,

(iii) are designed so as to direct the spatial positioning and/ororientation of bound molecules thereby facilitating crystallizationthereof, and

(iv) are designed so as to provide structural rigidity to boundmolecules thereby facilitating crystallization thereof.

Aside from enabling crystallization and subsequent atomic structuredetermination of previously uncharacterized molecules, the capacity ofthe present invention to multimerize and/or purify amolecule-of-interest can be advantageously applied in various biomedicalfields including protein therapeutics, oral lumenal therapies forgastrointestinal diseases and self-adjuvanting or subunit vaccines.

In addition, crystallization of macromolecule pharmaceuticals, and inparticular proteins, can be used to streamline manufacturing processes,as in the case with small-molecule drugs. Since a crystal is the mostconcentrated possible form of a protein, crystallization can bebeneficial for drugs, such as antibodies, which require high doses atthe delivery site. In addition, since the rate of crystal dissolutiondepends on its morphology, size, and the presence of excipients,crystalline proteins may also serve as a convenient carrier-free slowrelease dosage form (insulin is a good example). Finally, the stabilityof proteins in crystalline form is higher than that of correspondingsoluble or amorphous materials and, as such, crystallization can be usedto greatly increase the shelf life of a drug product.

Macromolecular crystals generated according to the teachings of thepresent invention also find important uses as catalysts, adsorbents,biosensors and chiral chromatographic media. These may also be employedin environmental applications, including, for example, the destructionof nerve agents, for bioremediation and civil defense.

In addition to the above, the present invention provides methods ofprotein purification via crystal formation.

As described hereinabove, suitable GPCR-binding domains of arrestinmolecules can be used to bind GPCRs with high affinity and specify. SuchGPCR binding domains of arrestin molecules can therefore be used asaffinity ligands for purification of such GPCRs.

Thus, according to the present invention, there is provided a method ofpurifying a GPCR from a sample containing a GPCR.

The method of purifying a GPCR from a sample is effected by subjectingthe sample to affinity chromatography using a GPCR binding domain of anarrestin molecule.

All criteria described hereinabove regarding selection and/ormodification of a GPCR binding domain of an arrestin molecule suitableas a binding domain of a molecular linker are applicable to selectionand/or modification of a GPCR binding domain of an arrestin moleculesuitable as a GPCR binding region of an affinity ligand for thepresently described purification method. As is described in Example 10of the Examples section below GPCR binding domains of an arrestinmolecule corresponding to SEQ ID NO: 3 or SEQ ID NO: 4 can be used toefficiently bind various types of GPCRs with high specifity andaffinity, and thereby to efficiently purify various GPCRs regardless ofthe activation-phosphorylation state thereof.

Preferably the affinity ligand includes a purification tag forfacilitating attachment of the affinity ligand to an affinitychromatography matrix.

As is described in Example 10 of the Examples section below an affinityligand conjugated to a Strep-tag can be conveniently bound to anaffinity matrix to which core streptavidin is conjugated.

Alternately, as is further described in Example 10 of the Examplessection below an affinity ligand conjugated to core streptavidin can beconveniently bound to an affinity matrix to which a Strep-tag oriminobiotin is conjugated.

Suitable protocols for all phases of affinity chromatographypurification of molecules are widely available in the literature of theart (see, for example: Wilchek M. & Chaiken I., 2000. Methods Mol Biol147:1-6; Jack, G. W. Immunoaffinity chromatography. Mol Biotechnol 1,59-86; Narayanan S R., 1994. Journal of Chromatography A 658:237-258;Nisnevitch M. & Firer M A., 2001. J Biochem Biophys Methods 49:467-80;Janson J C. & Kristiansen T. in Packings and Stationary Phases inChromatography Techniques (ed. Unger, K. K.) 747 (Marcel Dekker, NewYork, 1990); Clonis, Y. D. in HPLC of Macromolecules A PracticalApproach 157 (IRL Press, Oxford, 1989); Nilsson J. et al., 1997. ProteinExpr Purif. 11:1-16).

Preferably, the present invention is used to purify a GPCR such asrhodopsin or a class A GPCR.

Preferably, the present invention is used to purify a class A GPCR suchas m2 muscarinic cholinergic receptor.

Additional objects, advantages and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771; and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below.

Example 1 Generation of Ordered Crystals of Molecules of Interest byComplexation Thereof with Non-Polypeptidic Molecular Linkers

In order to facilitate ordered crystallization and atomic structuredetermination of a molecule-of-interest, non-polypeptidic molecularlinkers were designed having the capacity to form a crystallizablemolecular complex with molecules of a molecule-of-interest and,preferably, with a metal atom.

Materials and Methods:

Molecular linkers are generated to facilitate ordered crystallization ofmolecules-of-interest having the following characteristics: (a) theability to homomultimerize molecules-of-interest in selected geometricconfigurations, thereby facilitating ordered crystallization ofmolecules-of-interest which do not naturally aggregate in configurationssuitable therefor; (b) sufficient conformational rigidity so as tofacilitate ordered crystallization or ordered assembly ofmolecules-of-interest lacking sufficient conformational rigiditytherefor; (c) sufficient hydrophilicity so as to facilitatesolubilization in polar solvents, and thereby crystallization, understandard crystallization-inducing conditions of molecules-of-interestlacking sufficient hydrophilicity therefor, (d) binding moietiesspecific for desired regions of molecules-of-interest, therebyfacilitating multimerization of the molecules-of-interest; and (e) theability to specifically bind a metal atom being capable of facilitating3D crystallographic analysis of molecules-of-interest by enablinggeneration of initial phases for X-ray diffraction crystallography. Amodular organization of such molecular linkers is schematized in FIG. 1a.

Such linkers may extend a binding moiety from a multimerization scaffoldvia a first chain of 1-3 carbon or oxygen atoms, representative examplesof which are depicted in FIG. 1 b. These chains preferably terminate ina functional group such as [—CO₂H], [—OH], [—NH₂], [—CO₂], [—CONH],[—O], [—OCO] or [—NHCO] which are used to attach, via conventionalester, amide or ether formation, a second chain of suitable length andgeometry so as to enable attachment of monomers of amolecule-of-interest to the multimerizing scaffold of the molecularlinker in the desired spatial configuration. Such chains preferablyinclude a molecular group, such as [—(CH₂CH₂O)₂—O—CH₂CH₂—] or[—(CH₂)₄—], to which is attached the binding moiety. Such chains possesssufficient conformational rigidity and/or hydrophilicity so as tofacilitate crystallization of molecules of a molecule-of-interestcomplexed therewith lacking such conformational rigidity and/orhydrophilicity, respectively.

Moieties specific for binding molecules of interest are preferablypolypeptides capable of directly or indirectly mediating specificrecognition of a molecule-of-interest, such as core streptavidin,peptide tags or antibodies. Alternatively, molecules such as [—N⁺(CH₃)₃]or [—CO(CF₃)] can be employed to specifically bind amolecule-of-interest. Binding of metal atoms to molecular linkers can beeffected via the use of molecular linkers comprising multimerizationscaffolds based on molecules, such as porphyrin or hydroxime, which canbind metal atoms such as Pt²⁺, Ni²⁺, Cu²⁺ or Co²⁺.

Examples of molecular linkers capable of forming a crystallizablemolecular complex with a molecule-of-interest and specifically binding ametal atom include, for example, porphyrin-based molecular linkers(FIGS. 2 a and 2 b, respectively) or hydroxime-based molecular linkers(FIG. 3).

Thus, the molecular linkers of the present invention form molecularcomplexes with molecules of a molecule-of-interest being positioned in aselected spatial geometry facilitating crystallization thereof. Suchmolecular linkers further facilitate crystallographic analysis of amolecule-of-interest by incorporating within the crystallizablemolecular complex a metal atom used to generate initial phases duringX-ray crystallography.

Example 2 Chemical Synthesis of Porphyrin-Based Molecular Linkers

As described in Example 1, porphyrin-based molecular linkers can beemployed to facilitate crystallization of molecules of interest bymultimerizing these within substantially conformationally rigid and/orhydrophobic crystallizable molecular complexes. Such linkers furtherfacilitate determination of the atomic structure of molecules ofinterest by incorporating a platinum atom which can be employed togenerate initial phases during X-ray crystallographic analysis ofcrystals of such molecular complexes.

Synthetic procedures to generate crystallizable molecular complexes withporphyrin-based molecular linkers are depicted in FIGS. 4 a and 4 b.

The steps involved in synthetic processes to generate a porphyrin-basedmolecular linker,5,15-Di(2,6-di(ethoxycarbonymethoxy))porphyrinato-platinum (FIG. 4 b,Product No. 4), and the attachment of various molecular spacers/bindingdomains thereto are outlined below:

Materials and Methods:

Synthesis of 5,15-Di(2,6-di(ethoxycarbonymethoxy))porphyrin (Product No.3): Dipyrrolmethane (280 mg, 1.9 mmol) and2,6-di(ethoxycarbonymethoxy)-benzaldehyde (590 mg, 1.9 mmol) weredissolved in dichloromethane (300 ml) and purged with nitrogen. To thiswas added trifluoroacetic acid (75 ml, 1 mmol) and the solution wasstirred for 3 hours at room temperature. DDQ (450 mg, 2 mmol) was added,the mixture was stirred for 1 hour and neutralized with triethylamine(1.5 ml). The resultant mixture was purified by chromatography on asilica column, eluting with dichloromethane. The product was eluted as apurple band from the column and was obtained by evaporation of theeluate to give purple crystals (250 mg) of the product.

Synthesis of 5,15-Di(2,6-di(ethoxycarbonymethoxy))-porphyrinatoplatinum(Product No. 4, where X=OCH₂CO₂Et, R=H, n=2M=Pt): The product of theprevious reaction (250 mg) was dissolved in acetic acid (50 ml) and tothis was added dipotassium tetrachloroplatinate (112 mg) and the mixturewas refluxed for 10 min. The mixture was cooled and water (20 ml) wasadded. The product (350 mg) was filtered off and washed with 50% aqueousethanol.

Synthesis of 5,15-Di(2,6-di(ethoxycarbonymethoxy))porphyrinatoplatinum(Product No. 4, where X=OCH₂CO₂H, R=H, n=2, M=Pt): The product of theprevious step (350 mg) was suspended in 50% aqueous ethanol (50 ml)containing sodium hydroxide (500 mg) and refluxed for 3 hrs. The mixturewas then acidified drop-wise with concentrated HCl, to pH 1 and theproduct (280 mg) was filtered off.

Synthesis of5,15-Di(2,6-di((N-biotinylaminopropyl)amidocarbonymethoxy))porphyrinatoplatinum(Product No. 4, where X=OCH₂CO₂NH—(CH₂)₃NH(biotinyl), R=H, n=2, M=Pt):350 mg, 288 mmol of the product of the previous reaction was added to asolution of DCC (72 mg) in dioxane (100 ml) containing a catalyticamount of hydroxybenzotriazole (5 mg). 3-(biotinylamino)-propylamine (95mg, 320 mmol) was then added and the mixture was stirred overnight atroom temperature and filtered. The residue was washed with ethyl acetateand the filtrate was evaporated to give the crude product (605 mg). Theproduct was then be further purified by chromatography on a silica gelcolumn, eluting with ethyl acetate. Analogues of this compound aresynthesized similarly.

The number of moieties specific for the molecule-of-interest are givenby the index n. The steric encumbrance between such moieties determinethe geometry of the molecular scaffold, and thus the geometry of themolecule-of-interest-linker complex. The biotinyl moiety described abovecan be used, for example to bind any molecule-of-interest which has beenfused to streptavidin.

Example 3 Chemical Synthesis of a Hydroxime-Based Molecular Linker

A synthetic procedure to generate a hydroxime-based molecular linker forbinding two molecules of a molecule-of-interest, thereby generating acrystallizable molecular complex containing the molecule-of-interest, isdepicted in FIG. 5. Such a molecular linker further facilitatesdetermination of the crystal structure of the molecule-of-interest bychelating a copper atom which is employed to generate initial phasesduring X-ray crystallographic analysis of a crystal of the molecularcomplex.

Materials and Methods:

Synthesis of5-((2-trimethylammonium-ethoxy)digolyloxycarbonyl)-2-hydroxyacetophenoneoxime dichloride (intermediate No. 5 where X=CO.(OCH₂CH₂)₃N⁺Me₃, R′=Me,n=1): 5-Carboxy-2-hydroxyacetophenone oxime (1 g, 6 mmol) was dissolvedin dioxane (50 ml) containing DCC (0.95 g) and(2-trimethylammonium-ethoxy)-digol chloride (1.4 g) dissolved in dioxane(20 ml) and the mixture was stirred for 6 hours at room temperature. Themixture was filtered and the filtrate was evaporated to dryness. Theresidue was then dissolved in water and the product was purified by ionexchange chromatography on a Dowex cation exchange column and wasobtained as a viscous oil, on evaporation under high vacuum, as achloride salt.

Synthesis ofBis-[5((2-trimethylammonium-ethoxy)-digolyloxycarbonyl)-carboxy-2-hydroxyacetophenoneoxime] copper (II) chelate dichloride (Product No. 6, whereX=CO.(OCH₂CH₂)₃N⁺Me₃, R′=Me, n=1, M=Cu): 100 mg of the previous reactionproduct was dissolved in water (10 ml) and to this was added an aqueoussolution of copper (II) chloride (1.5 ml of 0.1M solution). The solutionwas stirred for 4 hours and the mixture was evaporated to dryness, underhigh vacuum, to yield the product (110 mg) as a green solid. Analoguesof this compound are synthesized similarly.

The quaternary ammonium moiety is employed to bind any molecule which isknown to bind positively charged groups via cation-π interactions, suchas acetylcholinesterase.

Example 4 Synthesis of a Non-Polypeptidic Molecular Linker withBiotinylated Moieties for Attachment of a Molecule-of-Interest Coupledto a Biotin-Binding Molecule

A modular system where a single type of molecular linker may bind arange of molecules of interest is highly desirable since this obviatesthe requirement of synthesizing a dedicated linker for eachmolecule-of-interest. This is effected, for polypeptides of interest,for example, by incorporating within the molecular linker and thepolypeptide of interest heterologous moieties, such as polypeptides,that specifically bind to each other.

Since one of the highest binding affinities known between any twonon-covalently associated molecules is that between core streptavidinand biotin, the use of such binding a pair is ideal for binding amolecule-of-interest to a molecular linker. Such a binding interactionserves to optimize crystallization of the molecule-of-interest since itfacilitates formation of a highly stable and rigid molecular complexwhich can be easily crystallized.

The synthetic process for linkage of a biotin moiety to aporphyrin-based molecular linker is outlined in FIG. 6 a and isperformed as follows:

Synthesis of 5,10,15,20-tetra-(3-ethoxycarbonyl)porphyrin (Product No.2), where X=OCH₂CO₂Et, n=1): Ethyl 3-formylbenzoate (5 g) and pyrrole (2g) were dissolved in chloroform (1 liter) and the solution was purgedwith nitrogen for 10 min. A solution of BF₃.Et₂O (4 ml of 2.5Msolution). After 1 hour chloranil (5.4 g) was added and the mixture wasrefluxed for 1 hour. The mixture was cooled to room temperature and 1equivalent of triethylamine was added. The solution was evaporated todryness to give the crude product, which was washed with methanol threetimes. The product remained as a purple solid (1.43 g). The product wasthen elaborated, analogously to the method described above for synthesisof porphyrin-based molecular linkers, into further examples of theinvention.

Example 5 Synthesis of a Hydroxime-Based Molecular Linker withTrimethylammonium Moieties for Attachment of Molecules of aMolecule-of-Interest

In order to bind molecules of a molecule-of-interest in the desiredspatial configuration within a crystallizable molecular complex amolecular linker, according to the method of the present invention, mustbe of a suitable dimension and geometry.

Such positioning of a molecule-of-interest within a crystallizablemolecular complex is effected, for example, by employing molecularlinkers with a hydroxime-based multimerization scaffold, as describedabove, to which molecules of a molecule-of-interest are attached viatrimethylammonium moieties. As well as allowing binding of molecules ofinterest to a molecular linker without steric hindrance,trimethylammonium, being of substantial hydrophilicity andconformational rigidity, further facilitates solubilization andcrystallization, respectively, of the molecular complex.

The chemical attachment of trimethylammonium to a hydroxime-basedmolecular linker is depicted in FIG. 6 b. As described above, inclusionof a metal atom within the hydroxime-based molecular linker facilitatesdetermination of the atomic structure of the molecule-of-interest byproviding initial phases during X-ray crystallographic analysis of acrystal of a molecular complex including a molecule-of-interest.

Example 6 Crystallizable Molecular Complexes Comprising a MutagenesisPolypeptide of Interest and a Heterologous Molecular Linker

Mutagenesis of a polypeptide of interest is employed so as to optimizethe crystallizability of a molecular complex formed by a linkertherewith.

The polypeptide of interest is mutagenized in order to adjust the stericfit between the molecular linker and the molecules of the polypeptide ofinterest. Such an adjustment is employed in order to optimize the numberand/or physico-chemical characteristics of the crystal contacts of thecrystallizable molecular complex formed by association of molecules ofthe polypeptide of interest with the molecular linker. Additionally,selected residues of the polypeptide of interest are mutagenized inorder to optimize the solubility and/or rigidity of the crystallizablemolecular complex formed by association of molecules of the polypeptideof interest with the molecular linker.

Acetylcholinesterase (AChE) and muscarinic acetylcholine receptor(mAChR) are molecules which are well characterized pharmacologically andAChE is known to crystallize in a series of well-characterized lattices.Thus, AChE is mutagenized so as to optimize its packing within amolecular linker when multimerized therewith.

Muscarinic acetylcholine receptor, whose 3D structure remains to bedetermined, is representative of a broad class of integral membraneproteins of great pharmacological importance. However, it is known tobind ligands possessing a similar structure to those binding AChE. Thusa modified molecular linker, based on the one employed forcrystallization of mutagenized AChE, as described above, is employed inorder to crystallize mAChR, an integral membrane protein.

Materials and Methods:

The molecule-of-interest is mutagenized via standard recombinanttechniques and is produced using a bacterial expression system. Thepurified protein is solubilized in a monodisperse solution according tostandard crystallization procedures available in the literature. To thissolution, a suitable amount of molecular linker is added. A 5 microliteraliquot of this molecular linker solution is added to 5 microliters ofmother solution on a siliconized glass coverslip (18-22 mm diameter).The coverslip is placed over a well containing a solution buffered atthe appropriate pH and adjusted to the optimal concentration ofprecipitants (e.g. PEG 5000 or ammonium sulfate). The drop is allowed toequilibrate at the appropriate temperature (e.g. 20° C.) for an amountof time necessary for the crystal to form.

Example 7 Crystallization of a Molecule-of-Interest by Complexation witha Molecular Linker Composed of a Homomultimerizing Molecule Conjugatedto a Modular Recognition Domain Specific for a Molecule-of-Interest

One of the most versatile, convenient and specific means of specificallybinding a molecule-of-interest is via antibodies.

Therefore, molecular linkers were designed consisting of a chimericpolypeptide composed of fused scFv, core streptavidin and histidine tagsegments, as depicted schematically in FIG. 7 a. Such single-chainFv-core streptavidin chimeric polypeptides and polypeptides includinghistidine tags have been previously described (Ladner, R. C. et al.,U.S. Pat. No. 4,946,778) and (Sheibani N., 1999. Prep BiochemBiotechnol. 29(1):77), respectively. The relative positions of thesingle-chain Fv molecule and the core streptavidin segments can also beinverted. The peptide sequences GSAA (SEQ ID NO: 1) and GS (SEQ ID NO:2) are inserted between the V_(L) and core streptavidin, and between thecore streptavidin and the His-tag domains, respectively, so as toprovide the required flexibility for appropriate folding of the fusionprotein.

Optionally, association of a metal atom with the crystallizablemolecular complex is effected via the use of a second chimericpolypeptide comprising Strep-tag, metal atom-binding and purificationtag segments, as depicted in FIG. 7 b. The Strep-tag domain of thischimera serves to bind the core streptavidin domain of the corestreptavidin-containing chimera described hereinabove and thus serves toassociate the molecule-of-interest with a metal atom binding molecule.Binding of the metal atom to the metal atom binding domain is effectedeither prior to, concomitantly or following the binding steps describedabove. Furthermore, the purification tag of the metal atom bindingchimera can be employed to perform the same functions as thepurification tag comprised in the core streptavidin-containing chimeradescribed above. The conformation of a tetramerized complex obtainedusing the above-described system is depicted in FIG. 8.

Such a molecular linker thus binds a molecule-of-interest via its scFvdomain, tetramerizes via its core streptavidin domain and can be easilyidentified by immunoblotting analysis or purified by affinitychromatography, either prior to or following binding of amolecule-of-interest, via its purification tag domain.

One advantage of utilizing streptavidin as the core of molecularlinkers, is that extensive literature exists for the expression andpurification of streptavidin itself (Wu S C. et al., 2002. ProteinExpression and Purification 24:348-356; Gallizia A. et al., 1998.Protein Expression and Purification 14:192-196) and of streptavidinfusion proteins (Sano T. & Cantor C R. 2000. Methods Enzymol.326:305-11). Smaller and more stable streptavidins than the native formhave been produced recombinantly (Sano T. et al., 1993. Journal ofBiological Chemistry 270:28204-28209) and the gene sequence has beenoptimized for expression in E. coli (Thompson L D. & Weber P C., 1993.Gene 136:243-6). The tetramer of these smaller “cores” displays enhancedstability under denaturing conditions, and their biotin binding sitesappear to be more accessible. A small core size is also preferable, asit helps to keep the size of the final polypeptidic molecular linker toa minimum, making the scaffold easier and cheaper to produce and purify.Smaller molecular linkers may be advantageous since, as a rule of thumb,a smaller and tightly packed multimerization scaffold will introduceless disorder in the final crystallization complex, thus ensuringoptimal ordering of crystals.

Crystallization of a molecule-of-interest using the above-describedmolecular linkers is achieved as follows:

The chimeric polypeptide described above is produced in a first step viastandard recombinant DNA, protein expression and protein purificationtechniques. In a second step, the molecule-of-interest is crystallizedwithin a crystallizable molecular complex formed by tetramerization ofthe chimera via core streptavidin, thereby generating a molecularlinker, and by binding of molecules of the molecule-of-interest to thescFv domains of the molecular linker.

The order in which these various non-covalent binding steps are effectedcan be essentially shuffled at will since these involve biologicalinteractions occurring under similar physiological conditions. Asdiscussed above, incorporation of a metal atom into a molecular complexcontaining a molecule-of-interest serves to facilitate solution of the3D atomic structure of the molecule-of-interest.

The scheme outlined hereinabove for crystallization of amolecule-of-interest is highly modular and flexible and the componentsthereof are interchangeable while retaining the basic functionalitiesrequired for formation of a crystallizable molecular complex. Forexample, the molecule-of-interest-specific scFv domain is exchangeablewith any other molecule specifically binding the molecule-of-interest.One such example is a toxin specific for a membrane receptor, asdescribed in the embodiments of the present invention. This is effectedby employing the genetic sequence encoding the toxin instead of that ofthe scFv during the recombinant DNA manipulation phase of thiscrystallization method. Similarly, the metal atom binding segment of thechimera described above is exchangeable, via chemical synthesis, with anon-polypeptidic metal chelating molecule, such as porphyrin orhydroxime described in Examples 4 and 5, respectively. When employingappropriate combinations of auxiliary functional domains within themolecular linker, the core streptavidin domain segment of the molecularlinker is exchangeable with any other suitable homomultimerizingmolecule.

An alternative method for association of a metal atom with thecrystallizable molecular complexes of the present invention involves theuse of a molecular linker composed of a single type of molecule whichincludes the metal atom binding segment as well as themolecule-of-interest-binding, homomultimerizing and purification tagsegments. This is effected, for example, via a chimeric polypeptideincluding all these functional segments.

Thus, such molecular linkers can be employed to facilitatecrystallization and 3D atomic structure determination of a moleculewhich can be bound by an antibody.

Example 8 Generation of Ordered Crystals of a PolypeptidicMolecule-of-Interest Via Expression as a Fusion Chimera with aHeterologous Homomultimerization Domain

In order to crystallize a polypeptidic molecule-of interest, themolecule-of-interest is expressed as a fusion chimera with apurification tag, such as an epitope tag, which is specifically bound bya purification tag-binding molecule utilized as the molecule-of-interestbinding moiety of the molecular linker.

Such a crystallization system presents the advantage of enabling asingle molecular linker to facilitate the crystallization of anypolypeptide-of-interest, modified as described above.

All alternatives described in Example 7 above pertaining to functionalsegments of molecular linkers, and to methods of including metal atomsin crystallizable complexes are applicable to the presently disclosedmethod.

Production of a chimeric polypeptide comprising the molecule-of-interestand the tag is effected by cloning nucleic acid sequences encoding themolecule-of-interest into a bacterial expression vector which comprisesa nucleic acid sequence encoding the tag, and which is configured toexpress the molecule-of-interest and the tag in-frame as a fusionprotein.

Suitable bacterial strains are transformed with the expression vector,and recombinant chimera produced by transformants is recovered usingstandard recombinant protein technology, and is crystallized usingstandard crystallization conditions for X-ray crystallography.

Thus, this method provides a means of facilitating the crystallizationand crystallographic analysis of a broad range of polypeptides ofinterest conjugated to a heterologous molecule via a single type ofmolecular linker.

Example 9 Generation of Crystals of G Protein Coupled Receptors Suitablefor Determination of Three Dimensional Atomic Structure Thereof

A very large number of human diseases are associated with G proteincoupled receptor disfunction, as illustrated by the fact that Gprotein-coupled receptors constitute the most prominent family of drugtargets, as described above. Nevertheless, pharmacological treatment ofdiseases associated with GPCRs remains suboptimal, however. Thus, thereis a very great need for novel GPCR specific drugs. One way to generatesuch drugs would be to elucidate the 3D atomic structure of GPCRs athigh resolution so as to enable the rational design of pharmacologicalagents capable of having a desired regulatory effect on the activity ofsuch receptors. However, prior art methods cannot be used to efficientlygenerate crystals of membrane proteins such as GPCRs, which crystalsbeing suitable for determining the 3D atomic structure of such receptorsat high resolution. In order to fulfill this important need, the presentinventors have designed molecular linkers capable of being used togenerate highly ordered, X-ray crystallography grade crystals of Gprotein coupled receptors suitable for X-ray crystallographic analysisof the 3D atomic structure of such receptors as follows.

Background:

Streptavidin: Streptavidin is a 159 amino acid residue protein producedby Streptomyces avidinii that binds up to four molecules of biotin withultra-high affinity (K_(d)˜10⁻¹⁵ M; Green N M., 1990. Methods inEnzymology 184:51-67), to form an ultra-stable homotetramer that doesnot dissociate even in the presence of 6 M urea (Kurzban G P., 1991. JBiol Chem. 266, 14470-14477). The crystallographic structure of corestreptavidin illustrates that each streptavidin monomer folds into aneight-stranded antiparallel β-barrel, with the biotin binding site builtby residues of the barrel itself and a loop of an adjacent subunit toform a very stable dimer (Freitag S. et al., 1997. Protein Science6:1157-1166). Extensive intersubunit contacts between the dimers giverise to the final tetrameric structure having tight quaternary assemblyand fixed geometry (Green N M., 1990. Methods in Enzymology 184:51-67).

Another advantage of using streptavidin as the core of a molecularlinker, is that extensive literature exists for the expression andpurification of streptavidin itself (Wu S C. et al., 2002. ProteinExpression and Purification 24:348-356; Gallizia A. et al., 1998.Protein Expression and Purification 14:192-196), and of streptavidinfusion proteins (Sano T. & Cantor C R. 2000. Methods Enzymol.326:305-11). Smaller and more stable streptavidins than the native formhave been produced recombinantly (Sano T. et al., 1993. Journal ofBiological Chemistry 270:28204-28209) and the gene sequence has beenoptimized for expression in E. coli (Thompson L D. & Weber P C., 1993.Gene 136:243-6). The tetramer of these smaller cores displays enhancedstability under denaturing conditions, and their biotin binding sitesappear to be more accessible. A small core size is also preferable, asit helps to keep the size of the final polypeptidic molecular linker toa minimum, making the scaffold easier and cheaper to produce and purify.Smaller molecular linkers may be advantageous since, as a rule of thumb,a smaller and tightly packed multimerization scaffolds will introduceless disorder in the final GPCR-linker complex, thus ensuring higherquality crystals.

Arrestins: The arrestin family consists of visual arrestin (v-arrestin,S-arrestin), cone-arrestin, β-arrestin (β-arrestin-1 and arrestin-2),and β-arrestin-2 (arrestin-3). V- and cone-arrestins are exclusivelyexpressed in rod and cone photoreceptors, respectively, and are highlyspecialized to bind specifically to rhodopsin, or cone cell pigments.The two closely related β-arrestins are ubiquitously expressed and areresponsible for the termination of the primary signaling event for most,if not all, class I (rhodopsin-like) GPCRs.

At the sequence level, visual arrestin is 60% identical to theβ-arrestins, which show 78% sequence identity between themselves. Thethree dimensional structure of v-arrestin (Hirsch J A. et al., 1999.Cell 97:257-69; Granzin, J. et al., 1998. Nature 391:918-21) and ofβ-arrestin (Han M. et al., 2001. Structure (Camb) 9:869-80) have beensolved and reported in the literature.

Arrestins bind with subnanomolar affinities (Gurevich V V. et al., 1995.Journal of Biological Chemistry 270:720-731) exclusively toagonist-activated GPCRs that have been phosphorylated by Gprotein-coupled receptor kinases (GRKs) on serine and threonine residueslocated in the third intracellular loop or carboxyl terminal tail(Gurevich V V. & Benovic J L., 1992. Journal of Biological Chemistry267:21919-21923; Lohse M. et al., 1992. J Biol Chem. 267:8558-8564;Lohse M J. et al., 1990. Science 248:1547-50). The association of asingle arrestin with a GRK-phosphorylated receptor uncouples thereceptor from its cognate G protein, resulting in the termination ofGPCR signaling, a process termed desensitization (Gurevich V V. &Benovic J L., 1992. Journal of Biological Chemistry 267:21919-21923;Lohse M. et al., 1992. J Biol Chem. 267:8558-8564; Lohse M J. et al.,1990. Science 248:1547-50; Pippig S. et al., 1993. Journal of BiologicalChemistry 268:3201-3208; Attramadal H. et al., 1992. J Biol Chem.267:17882-17890). In the case of β-arrestins, these molecules thentarget desensitized receptors to clathrin-coated pits for endocytosis byfunctioning as adaptor proteins that link the receptor to components ofthe endocytic machinery such as AP-2 and clathrin (Goodman, O B Jr. etal., 1996. Nature 383:447-50; Laporte S A. et al., 1999. Proc Natl AcadSci USA. 96:3712-3717; Laporte S A. et al., 2000. J Biol Chem.275:23120-23126; Ferguson S S G. et al., 1996. Science 271:363-366). Theinternalized receptors are dephosphorylated in endosomes and recycledback to the cell surface fully resensitized (Zhang L. et al., 1997. JBiol Chem. 272:14762-8; Oakley R H. et al., 1999. J Biol Chem.274:32248-57; Krueger K M. et al., 1997. J Biol Chem 272:5-8).

The overall structures of β-arrestins and v-arrestin share many similarfeatures: all are elongated molecules with a central polar core built bya network of charge-charge interactions (amino acid residues 1-8, 30,175-176, 296, 303 and 382; where the numbering follows the sequence ofv-arrestin) flanked by the N (amino acid residues 8-180) domain, Cdomain (amino acid residues 188-362) and a C tail (amino acid residues372-404) that tightly interacts with the two domains and with the Nterminus. Residues 98-108 in the N-domain form a cationic amphipathicα-helix that might serve as a reversible membrane anchor. Structuralvariations between arrestins are mostly found in surface loops. Analysisof β-arrestin and v-arrestin structures has shown that such arrestinsare characterized by a very similar overall structure (Han M. et al.,2001. Structure (Camb) 9:869-80). The loop regions that vary betweenβ-arrestin and v-arrestin also vary between different crystal forms ofthe same protein, reflecting the intrinsic flexibility of those regionsrather than inherent structural differences between the two arrestins,as can be seen from the distribution of B factors. The crystalstructures of v-arrestin and of β-arrestin analyzed represent theirrespective inactive basal states, where the polar core is intact.

It has been shown that the predominant region of receptor binding inv-arrestin is contained within amino acid residues 90-140. A portion ofthis region (amino acid residues 95-140) expressed as a fusion proteinwith glutathione S-transferase has been shown to be capable of bindingto rhodopsin regardless of the activation or phosphorylation state ofthe receptor (Smith W C. et al., 1999. Biochemistry 38:2752-61).Mutations disrupting the polar core such as the v-arrestin mutant R175E,promote phosphorylation-independent binding of arrestin to the receptor(Gurevich V V. & Benovic J L. Molecular Pharmacology 51:161-169;GrayKeller M P. et al., 1997. Biochemistry 36:7058-7063).Segment-swapping experiments between visual and non-visual arrestinshave demonstrated that substituting amino acid residues 50-90 ofv-arrestin with the equivalent element of β-arrestin (amino acidresidues 46-86) can switch the binding specificity of v-arrestin to highaffinity binding of activation-phosphorylated m2 muscarinic cholinergicreceptor (P-m2 mAchR*) while losing the affinity foractivation-phosphorylated rhodopsin (P—Rh*; Han M. et al., 2001.Structure (Camb) 9:869-80). Remarkably, the single amino acid mutationV90S was shown to eliminate this difference, permitting v-arrestin tobind P-m2 mAchR* with similar affinity as β-arrestin without significantconcurrent loss of its affinity to P—Rh*. In addition, elimination ofthe hydrophobic side chains of residues 11-13 was observed to disruptthe interaction between the N-domain and the amphipathic α-helix, andenhances phosphorylation-independent binding of arrestin (VishnivetskiyS A. et al., 2000. J Biol Chem. 275:41049-41057).

These truncation and deletion studies point to the N-terminal domain asthe primary domain of interaction—the truncated N-domain of arrestinbinds to P-m2 mAchR with a K_(d)=2 nM (Gurevich V V. et al., 1995.Journal of Biological Chemistry 270:720-731). Additional data also pointto the C-domain as playing a significant role in receptor binding sincea truncated form of arrestin in which just a short C-terminal region isremoved displays a K_(d)=1 nM (Gurevich V V. et al., 1995. Journal ofBiological Chemistry 270:720-731).

The evidence accumulated so far suggest two possible mechanismspromoting receptor-arrestin interaction that are independent of thespecific GPCR subtype. One mechanism is linked to the polar core, wherecritical salt bridges keep arrestin in its basal state (Hirsch J A. etal., 1999. Cell 97:257-69). An activation-phosphorylated GPCR wouldinteract with arrestin, thereby disrupting the polar core and triggeringthe conformational changes required for high-affinity receptor binding.A second general mechanism can be derived from structural andmutagenesis data, whereby receptor binding is triggered and/or enhancedby the membrane translocation of arrestin's amphipathic α-helix I (HanM. et al., 2001. Structure (Camb) 9:869-80).

Materials and Methods:

The above-described data relating to streptavidin indicates that corestreptavidin can be used to generate molecular linkers having a highlystable and rigid predetermined quaternary structure and geometrysuitable for optimally facilitating crystallization of crystallizationcomplexes. The above-described data relating to arrestins indicates thata polypeptide composed of amino acid residues 11-190 of humanbeta-arrestin-1a with mutation R169E (SEQ ID NO: 3; FIG. 9 a), or apolypeptide composed of amino acid residues 11-370 of humanbeta-arrestin-1a with mutation R169E (SEQ ID NO: 4; FIG. 9 b) can serveas ligands capable of binding different classes of GPCRs with highaffinity and specificity regardless of the phosphorylation/activationstate thereof. Mutation R169E in human beta-arrestin-1a is homologous tothe above-described R175E mutation in v-arrestin, as shown by publishedamino acid sequence comparisons (Han M. et al., 2001. Structure (Camb)9:869-80; Hirsch J A. et al., 1999. Cell 97:257-69). Mutation R169E thusenables binding of GPCRs independently of the activation-phosphorylationstate thereof. There is a serine residue located at position 86 inwild-type human beta-arrestin-1a which corresponds to mutation V90S inv-arrestin as shown by the aforementioned published amino acid sequencecomparisons. As described hereinabove, the presence of a serine residueat this position confers the capacity to bind multiple types of GPCRs.Thus, the polypeptides corresponding to SEQ ID NOs: 3 and 4 have thecapacity to bind multiple types of GPCRs as well as the capacity to bindGPCRs independently of the activation-phosphorylation state thereof.Thus, molecular linkers were designed incorporating a streptavidin basedcore and arrestin based GPCR binding portions.

Streptavidin-arrestin chimera based molecular linkers: Two polypeptidicmolecular linkers for generation of X-ray crystallography grade crystalsof molecular linker-GPCR complexes were designed. The first linker (SEQID NO: 5; FIG. 10 a) is composed of a chimeric protein consisting of theN- to C-terminal segments; T7 tag, core streptavidin, the peptide linkerGSAA (SEQ ID NO: 1), and the above-described human beta-arrestin-1aderived polypeptide set forth in SEQ ID NO: 3. The second linker (SEQ IDNO: 6; FIG. 10 b) is composed of a chimeric protein consisting of the N-to C-terminal segments; T7 tag, core streptavidin, the peptide linkerGSAA (SEQ ID NO: 1), and the above-described human beta-arrestin-1asegment set forth in SEQ ID NO: 4.

These molecular linkers can be conjugated to a metal atom viabiotinylated porphyrin synthesized, as described above. Molecularlinkers having streptavidin cores can adopt a highly stable and rigidpredetermined quaternary structure and geometry suitable for optimallyfacilitating crystallization of crystallization complexes, and bind withhigh specificity and affinity the largest possible set of differentGPCRs.

Streptavidin-metallothionein chimera/arrestin-Strep-tag chimera basedmolecular linkers: Polypeptidic molecular linkers for generation ofX-ray crystallography grade crystals of molecular linker-GPCR complexeswere designed using a system of two polypeptide chimeras. One chimeraconsists of the N- to C-terminal segments; T7 tag, core streptavidin,and metallothionein. The other chimera consists of, the N- to C-terminalsegments; the above-described human beta-arrestin-1a derived polypeptideset forth in SEQ ID NO: 3 or SEQ ID NO: 4 and a Strep-tag. In thissystem, the arrestin comprising chimera is attached to the core of themolecular linker by specific binding of the Strep-tag, to which thearrestin derived polypeptide is fused, to the core streptavidincontained in the molecular linker. The metallothionein segment can beused to incorporate several heavy metal atoms such as Cd²⁺ in thecrystallization complex for providing initial phases for analysis ofX-ray crystal diffraction data.

Metallothionein-streptavidin fusion proteins are produced essentially aspreviously described in the literature, with minor modifications forincluding the T7 tag and for adjusting the length of the streptavidincore (Sano T. et al., 1999. Proc Natl Acad Sci USA. 89:1534-8).

The T7 tag was used in order to increase production of recombinantproteins and to facilitate their purification.

The availability of the 3D structures of all proteins employed in theconstruction of the above-described polypeptidic molecular linkers hasenabled modeling of the structure of such molecular linkers with asignificant degree of confidence.

Chimeric proteins are cloned in standard expression vectors forexpression of recombinant proteins in E. coli using standard recombinantDNA procedures on the basis of genomic DNA sequences, cDNA sequences orprotein sequences of arrestins and streptavidins available in public andprivate databases (e.g., GenBank, EMBL, PIR, NCBI Pubmed, etc).Sequences coding for the fusion protein are codon-optimized forexpression in E. coli (Thompson L D. & Weber P C., 1993. Gene136:243-6). Streptavidin fusion proteins are optimally designed andproduced with the streptavidin core at the N-terminus and are producedas inclusion bodies to maximize free biotin binding sites and refoldingas previously described (Sano T. & Cantor C R. 2000. Methods Enzymol.326:305-11). Introduction of the T7 tag at the N-terminus of thechimeric proteins increases expression thereof and permits easierpurification thereof (Gallizia A. et al., 1998. Protein Expression andPurification 14:192-196. Recombinant chimeras are purified frombacterial inclusion bodies using standard techniques and T7 tag specificaffinity chromatography. The purified molecular linkers are thenindividually mixed with different types of GPCRs at stoichiometricratios, and under physiological conditions suitable for enabling complexformation therebetween. Formed complexes are subsequently subjected tocrystallization inducing conditions.

For a one-step purification/molecular linker complexation procedure,fusion proteins containing core streptavidin, or molecular complexescontaining such fusion proteins, are bound to affinity chromatographycolumns with matrices conjugated to streptavidin specific ligands, andare directly eluted from such columns using biotinylated molecularlinker, such as biotinylated porphyrin (described above).

The monodispersity and second virial coefficient of solutions containingmolecular linkers, GPCRs, and complexes comprising molecular linkersand/or GPCRs are monitored via light scattering techniques so as toselect optimal preparations thereof for crystallization (Curtis R A. etal., 2001. Journal of Physical Chemistry B 105:2445-2452; Ruppert S. etal., 2001. Biotechnology Progress 17:182-187; Hitscherich C. et al.,2000. Protein Science 9:1559-1566).

Results:

With each of the above-described types of molecular linkers, differenttypes of GPCRs are efficiently crystallized conjugated to heavy metalatoms suitable for generating initial phases for X-ray crystallographicanalysis of 3D atomic structure. Such crystals are highly ordered, X-raycrystallography grade, crystals.

Conclusion: The above-described GPCR crystallization method can be usedto generate highly purified, highly ordered, X-ray crystallography gradecrystals of numerous classes of GPCRs, regardless of theactivation/phosphorylation state thereof, suitable for determining the3D atomic structure of such GPCRs. The present method is superior to allprior art methods, since prior art methods cannot be used to efficientlygenerate highly ordered crystals of different types of GPCRs.

Example 10 Efficient Purification of Different Classes of CorrectlyFolded G Protein-Coupled Receptors Via Arrestin Based AffinityChromatography

As described in the previous Example, there is a vital need for novelGPCR targeting drugs. In order to provide the data required forproducing such drugs, pharmacological, biochemical, and structuralstudies must be performed on GPCRs. Such studies require significantquantities of highly purified, correctly folded GPCRs. There istherefore a need for methods of producing large quantities of varioustypes of correctly folded GPCRs. Various prior art approaches have beenattempted for purifying GPCRs. One approach, has attempted isolating andpurifying GPCRs from primary tissues. Another approach has attempted toisolate and purify GPCRs via expression of such molecules as recombinantproteins in heterologous systems. However, all prior art approaches areunsatisfactory for producing satisfactory yields of correctly foldedGPCRs due to the low natural abundance of GPCRs in primary tissues, anddue to the lack of a suitable method of purifying GPCRs, membraneproteins whose correct folding is highly dependent on the membranalenvironment, in the correctly folded state. Furthermore, prior artapproaches cannot be used to efficiently purify multiple GPCR types. Forexample, purification tag based purification systems cannot discriminatebetween folded and unfolded states of tagged proteins, and furthermoreare restricted by the requirement that the tag be accessible on thesurface of the protein, and not buried within the protein. Affinitypurification techniques based on monoclonal antibodies or specificreceptor ligands require cumbersome testing and preparation, areexpensive, and are typically dedicated to a single type of targetmolecule. Thus, all prior art approaches have failed to provide anadequate solution for efficient production of purified, correctlyfolded, GPCRs of various types. In order to fulfill this important need,the present inventors have devised a novel and improved method ofisolating GPCRs as follows.

Materials and Methods:

The capacity of the above-described arrestin-derived polypeptides (SEQID NOs: 3 and 4) to bind numerous classes of GPCRs regardless of theactivation-phosphorylation state thereof indicates that suchpolypeptides constitute ideal capture ligands for affinitychromatography of a wide range of GPCRs. Such forms of arrestin are usedfor affinity chromatography purification of GPCRs as follows.

Each of the above-described GPCR-binding human beta-arrestin-1a derivedpolypeptides (SEQ ID NOs: 3 and 4) is synthesized via standardrecombinant protein production techniques, and is individually coupledto a suitable affinity purification support matrix such as an agarose,polyacrylamide, silica, cellulose or dextran matrix (Wilchek M. &Chaiken I., 2000. Methods Mol Biol 147:1-6; Jack, G W., 1994. MolBiotechnol. 1:59-86; Narayanan S R., 1994. Journal of Chromatography A658:237-258; Nisnevitch M. & Firer M A., 2001. J Biochem Biophys Methods49:467-80; Janson J C. & Kristiansen T. in Packings and StationaryPhases in Chromatography Techniques (ed. Unger, K. K.) 747 (MarcelDekker, New York, 1990)).

The GPCR-binding polypeptides are coupled to the support matrixcovalently and in an orientation specific manner via a standard couplingreaction (see, for example: Wilchek M. & Chaiken I., 2000. Methods MolBiol 147:1-6; Jack G W., 1994. Mol Biotechnol. 1:59-86; Narayanan S R.,1994. Journal of Chromatography A 658:237-258; Nisnevitch M. & Firer MA., 2001. J Biochem Biophys Methods 49:467-80; Clonis Y D. in HPLC ofMacromolecules A Practical Approach 157 (IRL Press, Oxford, 1989)).

Alternatively, GPCR-binding polypeptides are produced fused to aStrep-tag (Schmidt T G M. et al., 1996. Journal of Molecular Biology255:753-766; Skerra A. & Schmidt T G M., 1999. Biomolecular Engineering16:79-86), as previously described (Nilsson J. et al., 1997. ProteinExpr Purif. 11:1-16), and is coupled to a support matrix conjugated tostreptavidin.

As a further alternative, the arrestin segment is produced fused to anN-terminal core streptavidin moiety and is a coupled to a support matrixconjugated with Strep-tag peptide or iminobiotin (Sano T. et al., 1998.Journal of Chromatography B 715:85-91).

An affinity chromatography column is prepared using thearrestin-conjugated matrix, a sample containing a soluble GPCR isapplied to the column, the column is subjected to a cycle of washes forremoval of contaminants, and fractions are eluted using a suitablebuffer. Free GPCR is then eluted using a buffer containing a peptidethat specifically competes with GPCR for binding with arrestin (GurevichV V. et al., 1995. Journal of Biological Chemistry 270:720-731; Smith,W. C. et al., 1999. Biochemistry 38:2752; Raman D. et al., 1999.Biochemistry 38:5117-23; Bennett T A. et al., 2001. J Biol Chem.276:22453-60; Sternemarr R. et al., 1993. Journal of BiologicalChemistry 268:15640-15648); tagged arrestin-GPCR complex is eluted usinga standard buffer specific for uncoupling the tag from itsmatrix-conjugated ligand (Nilsson J. et al., 1997. Protein Expr Purif.11:1-16); or streptavidin-arrestin fusion protein is eluted with biotin,or a biotinylated molecule, such as biotinylated porphyrin, as describedin the preceding Example, thereby enabling simultaneous purification andmolecular linker complexation thereof. Elution of GPCR as a complex withthe arrestin ligand is advantageous for obtaining correctly folded GPCRin high yield due to arrestin functioning as a stabilizing adjuvant tothe receptor preparation (Hulme E C. & Curtis C A., 1998. BiochemicalSociety Transactions 26:S361) Separation of GPCR from tagged arrestin isthen effected using the aforementioned peptide that specificallycompetes with the GPCR for binding with arrestin.

Purification of GPCR in eluted fractions is monitored via standard lightscattering techniques.

The above described procedure is repeated using different classes ofunmodified or suitably modified GPCRs using the same type of, or thesame suitably recycled, purification column.

Results:

Significant quantities of highly purified, correctly folded GPCRs ofnumerous classes are produced.

Conclusion: The above-described method of the present invention can beused conveniently and rapidly produce large quantities of highlypurified, correctly folded GPCRs of different classes. Such purifiedGPCRs can be used to obtain valuable information required for generatingnovel GPCR-targeting drugs. As such, the method of the present inventionis significantly superior to prior art methods which cannot be used toefficiently purify various types of correctly folded GPCRs insignificant quantities.

Example 11 Universal Molecular Linkers for Crystallization ofHistidine-Tagged Membrane Proteins

Solution of the 3D structure of membrane proteins, is crucial for therational design of drugs targeting such proteins. To date, X-raydiffraction analysis of highly ordered crystals comprising such proteinsremains the only way to solve the 3D atomic structure of such proteins.However, no prior art crystallization methods can be used to efficientlygenerate such crystals. In order to fulfill the critical need for suchmethods, the present inventors have devised universal molecular linkersfor crystallizing essentially any histidine tagged membrane protein.

Materials and Methods:

Crystallization via porphyrin-NTA-Ni²⁺ molecular linker: A porphyrinbased molecular linker comprisingN-(5-amino-1-carboxypentyl)imino-diacetic acid (NTA) groups issynthesized and is chelated to Ni²⁺ using standard chemical techniques.A schematic diagram of porphyrin-NTA-Ni²⁺ molecular linker is shown inFIG. 11. A sample containing a recombinant histidine tagged membraneprotein displaying an accessible histidine tag is generated usingstandard techniques (e.g., refer to Sheibani N., 1999. Prep BiochemBiotechnol. 29:77). The sample containing the histidine-tagged membraneprotein is reacted with porphyrin-NTA-Ni²⁺ in the appropriatestoichiometry and under suitable reaction conditions for formation ofcomplexes of porphyrin-NTA-Ni²⁺ and the histidine-tagged protein.Complexation occurs via association of the chelated nickel ion with thehistidine tag of the membrane protein. The complex is purified,dissolved in a suitable buffer, and is crystallized using standardcrystallization conditions.

The above described process is repeated using different histidine-taggedmembrane proteins.

Crystallization via anti histidine tag single-chain Fv-core streptavidinfusion protein molecular linker: In order to crystallize a membraneprotein-of-interest, a polypeptidic molecular linker composed of afusion protein comprising, from N- to C-terminal; anti histidine tagsingle chain Fv derived from monoclonal antibody 3D5 (Kaufmann, M. etal., 2002. J Mol Biol. 318. 135-47) and core streptavidin is generated.The recombinant single chain Fv-core streptavidin chimera is produced aspreviously described, with minor modifications (see, for example:Cloutier S M. et al., 2000. Molecular Immunology 37:1067-1077; Dubel S.et al., 1995. J Immunol Methods 178:201; Huston J S. et al., 1991.Methods in Enzymology 203:46; Kipriyanov S M. et al., 1995. HumAntibodies Hybridomas 6:93; Kipriyanov S M. et al., 1996. ProteinEngineering 9:203; Pearce L A. et al., 1997. Biochem Molec Biol Intl42:1179-1188). The membrane protein-of-interest is produced as arecombinant histidine tagged protein displaying an accessible histidinetag using standard techniques (e.g., refer to Sheibani N. 1999. PrepBiochem Biotechnol. 29:77). A sample containing the histidine-taggedmembrane protein-of-interest is reacted with the single chain Fv-corestreptavidin molecular linker in an appropriate stoichiometry undersuitable reaction conditions for formation of complexes of the molecularlinker and the histidine-tagged protein (refer, for example to:Kaufmann, M. et al., 2002. J Mol Biol. 318. 135-47). The complex ispurified, dissolved in a suitable buffer, and is crystallized usingstandard crystallization conditions.

Results:

Highly ordered, X-ray crystallography grade crystals, each containing adifferent membrane protein, are efficiently generated using bothporphyrin-NTA and anti histidine tag single-chain Fv-core streptavidinbased molecular linkers.

Conclusions: The above-described molecular linkers can be used toefficiently generate different highly ordered, X-ray crystallographygrade crystals, each comprising a different membrane protein. Suchcrystals can be used to determine the 3D atomic structure of suchmembrane proteins. As such the method of the present invention issuperior to all prior art methods of generating membrane proteins sincethese cannot be used to efficiently generate highly ordered crystals ofmembrane proteins.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents, patent applicationsand sequences identified by their accession numbers mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent, patent application or sequence identified by theiraccession number was specifically and individually indicated to beincorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention.

1. A method of generating a crystal containing a molecule-of-interest,the method comprising: (a) contacting molecules of themolecule-of-interest with at least one type of heterologous molecularlinker being capable of interlinking at least two molecules of themolecule-of-interest to thereby form a crystallizable molecular complexof defined geometry; and (b) subjecting said crystallizable molecularcomplex to crystallization-inducing conditions, thereby generating thecrystal containing the molecule-of-interest.
 2. The method of claim 1,wherein said at least one type of heterologous molecular linker isselected such that said crystallizable molecular complex formed iscapable of generating a crystal selected from the group consisting of a2D crystal, a helical crystal and a 3D crystal.
 3. The method of claim1, wherein the molecule-of-interest is a polypeptide.
 4. The method ofclaim 3, wherein said polypeptide is a membrane protein.
 5. The methodof claim 4, wherein said membrane protein is a G protein coupledreceptor.
 6. The method of claim 5, wherein said G protein coupledreceptor is rhodopsin or is a class A G protein coupled receptor.
 7. Themethod of claim 6, wherein said class A G protein coupled receptor is m2muscarinic cholinergic receptor.
 8. The method of claim 1, wherein saidat least one type of heterologous molecular linker includes a region forspecifically binding the molecule-of-interest.
 9. The method of claim 8,wherein the molecule-of-interest is a G protein coupled receptor andwhereas said region for specifically binding the molecule-of-interestcomprises a molecule selected from the group consisting of at least aportion of an arrestin molecule, at least a portion of an arrestinmolecule having a mutation at an amino acid residue positioncorresponding to position 90 in bovine visual arrestin, at least aportion of an arrestin molecule having a mutation at an amino acidresidue position corresponding to position 175 in bovine visualarrestin, SEQ ID NO: 3, and SEQ ID NO:
 4. 10. The method of claim 9,wherein said at least a portion of an arrestin molecule is homologous toamino acid residues 11 to 190, or 11 to 370 of human beta-arrestin-1a.11. The method of claim 9, wherein said at least a portion of anarrestin molecule comprises a G protein coupled receptor-binding domainof said arrestin molecule.
 12. The method of claim 9, wherein saidmutation at an amino acid residue position corresponding to position 90in bovine visual arrestin is a mutation to a serine or threonineresidue.
 13. The method of claim 9, wherein said mutation at an aminoacid residue position corresponding to position 175 in bovine visualarrestin is a mutation to a glutamic acid or an asparagine residue. 14.The method of claim 9, wherein said G protein coupled receptor isrhodopsin or is a class A G protein coupled receptor.
 15. The method ofclaim 14, wherein said class A G protein coupled receptor is m2muscarinic cholinergic receptor.
 16. The method of claim 8, wherein themolecule-of-interest includes a histidine tag and whereas said regionfor specifically binding the molecule-of-interest comprises a nickel ionor an antibody specific for said histidine tag.
 17. The method of claim8, wherein the molecule-of-interest includes core streptavidin andwhereas said region for specifically binding the molecule-of-interestcomprises a biotin moiety or a Strep-tag.
 18. The method of claim 8,wherein the molecule-of-interest includes a biotin moiety or a Strep-tagand whereas said region for specifically binding themolecule-of-interest comprises core streptavidin.
 19. The method ofclaim 1, wherein the molecule-of-interest is a G protein coupledreceptor and whereas said at least one type of molecular linkercomprises a molecule selected from the group consisting of at least aportion of an arrestin molecule, at least a portion of an arrestinmolecule having a mutation at an amino acid residue positioncorresponding to position 90 in bovine visual arrestin, at least aportion of an arrestin molecule having a mutation at an amino acidresidue position corresponding to position 175 in bovine visualarrestin, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.20. The method of claim 19, wherein said at least a portion of anarrestin molecule is homologous to amino acid residues 11 to 190, or 11to 370 of human beta-arrestin-1a.
 21. The method of claim 9, whereinsaid at least a portion of an arrestin molecule comprises a G proteincoupled receptor-binding domain of said arrestin molecule.
 22. Themethod of claim 19, wherein said mutation at an amino acid residueposition corresponding to position 90 in bovine visual arrestin is amutation to a serine or threonine residue.
 23. The method of claim 19,wherein said mutation at an amino acid residue position corresponding toposition 175 in bovine visual arrestin is a mutation to a glutamic acidor an asparagine residue.
 24. The method of claim 19, wherein said Gprotein coupled receptor is rhodopsin or is a class A G protein coupledreceptor.
 25. The method of claim 24, wherein said class A G proteincoupled receptor is m2 muscarinic cholinergic receptor.
 26. The methodof claim 1 wherein said at least one type of heterologous molecularlinker includes at least two non-covalently bound subunits.
 27. Themethod of claim 26, wherein said at least two non-covalently boundsubunits comprise a first subunit comprising a homomultimerizing portionand a metal-binding portion, and a second subunit comprising a portionspecifically binding the molecule-of-interest, and a portionspecifically binding said first subunit.
 28. The method of claim 26,wherein said at least two non-covalently bound subunits comprise a firstsubunit comprising a homomultimerizing portion and a portionspecifically binding the molecule-of-interest, and a second subunitcomprising a metal-binding portion, and a portion specifically bindingsaid first subunit.
 29. The method of claim 1, wherein said at least onetype of heterologous molecular linker includes a molecule selected fromthe group consisting of a polycyclic molecule, a polydentate ligand, amacrobicyclic cryptand, a polypeptide and a metal.
 30. The method ofclaim 1, wherein said at least one type of heterologous molecular linkercomprises core streptavidin.
 31. The method of claim 1, wherein said atleast one type of heterologous molecular linker is selected so as todefine the spatial positioning and orientation of said at least twomolecules within said crystallizable molecular complex, therebyfacilitating crystallization of the molecule-of-interest.
 32. The methodof claim 1, wherein said at least one type of heterologous molecularlinker includes a hydrophilic region, said hydrophilic region being forfacilitating crystallization of the molecule-of-interest.
 33. The methodof claim 1, wherein said at least one type of heterologous molecularlinker includes a conformationally rigid region, said conformationallyrigid region being for facilitating crystallization of themolecule-of-interest.
 34. The method of claim 1, wherein said at leastone type of heterologous molecular linker includes a metal-bindingmoiety capable of specifically binding a metal atom, said metal atombeing capable of facilitating crystallographic analysis of the crystal.35. The method of claim 34, wherein said metal-binding moiety is a metalbinding protein.
 36. The method of claim 35, wherein said metal bindingprotein is metallothionein.
 37. The method of claim 1, wherein said atleast one type of heterologous molecular linker includes a region beingcapable of functioning as a purification tag, said purification tagbeing capable of facilitating purification of said crystallizablemolecular complex and/or of facilitating said interlinking at least twomolecules of the molecule-of-interest.
 38. The method of claim 37,wherein said region being capable of functioning as a purification tagis selected from the group consisting of a T7 tag, a histidine tag, aStrep-tag, core streptavidin, and biotin.
 39. The method of claim 1,wherein the molecule-of-interest includes a region being capable offunctioning as a purification tag, said purification tag being capableof facilitating purification of said crystallizable molecular complex,and/or of facilitating said interlinking at least two molecules of themolecule-of-interest.
 40. The method of claim 39, wherein said regionbeing capable of functioning as a purification tag is selected from thegroup consisting of a T7 tag, a histidine tag, a Strep-tag, corestreptavidin, and biotin.
 41. The method of claim 1, wherein themolecule-of-interest includes a metal-binding moiety capable ofspecifically binding a metal atom, said metal atom being capable offacilitating crystallographic analysis of the crystal.
 42. The method ofclaim 41, wherein said metal-binding moiety is a metal binding protein.43. The method of claim 42, wherein said metal binding protein ismetallothionein.
 44. A method of generating a crystal containing apolypeptide of interest, the method comprising: (a) providing a moleculeincluding the polypeptide of interest and a heterologous multimerizationdomain being capable of directing the homomultimerization of thepolypeptide of interest; (b) subjecting said molecule tohomomultimerization-inducing conditions, thereby forming acrystallizable molecular complex; and (c) subjecting said crystallizablemolecular complex to crystallization-inducing conditions, therebygenerating the crystal containing the polypeptide of interest.
 45. Themethod of claim 44, wherein (a) and (b) are effected concomitantly. 46.The method of claim 44, wherein said heterologous multimerization domainis selected such that said crystallizable molecular complex formed iscapable of generating a crystal selected from the group consisting of a2D crystal, a helical crystal and a 3D crystal.
 47. The method of claim44, wherein said heterologous multimerization domain includes ahydrophilic region, said hydrophilic region being for facilitatingcrystallization of the polypeptide of interest.
 48. The method of claim44, wherein said heterologous multimerization domain includes aconformationally rigid region, said conformationally rigid region beingfor facilitating crystallization of the polypeptide of interest.
 49. Themethod of claim 44, wherein said heterologous multimerization domain isselected so as to define the spatial positioning and orientation ofpolypeptides of the polypeptide of interest within said crystallizablemolecular complex, thereby facilitating crystallization of thepolypeptide of interest.
 50. The method of claim 44, wherein saidheterologous multimerization domain comprises core streptavidin.
 51. Themethod of claim 44, wherein the polypeptide of interest is a G proteincoupled receptor and whereas said heterologous multimerization domaincomprises a molecule selected from the group consisting of at least aportion of an arrestin molecule, at least a portion of an arrestinmolecule having a mutation at an amino acid residue positioncorresponding to position 90 in bovine visual arrestin, at least aportion of an arrestin molecule having a mutation at an amino acidresidue position corresponding to position 175 in bovine visualarrestin, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.52. The method of claim 51, wherein said at least a portion of anarrestin molecule is homologous to amino acid residues 11 to 190, or 11to 370 of human beta-arrestin-1a.
 53. The method of claim 52, whereinsaid at least a portion of an arrestin molecule comprises a G proteincoupled receptor-binding domain of said arrestin molecule.
 54. Themethod of claim 51, wherein said mutation at an amino acid residueposition corresponding to position 90 in bovine visual arrestin is amutation to a serine or threonine residue.
 55. The method of claim 51,wherein said mutation at an amino acid residue position corresponding toposition 175 in bovine visual arrestin is a mutation to a glutamic acidor an asparagine residue.
 56. The method of claim 5 1, wherein said Gprotein coupled receptor is rhodopsin or is a class A G protein coupledreceptor.
 57. The method of claim 56, wherein said class A G proteincoupled receptor is m2 muscarinic cholinergic receptor.
 58. The methodof claim 44, wherein the polypeptide of interest includes a histidinetag and whereas said heterologous multimerization domain comprises anickel ion or an antibody specific for said histidine tag.
 59. Themethod of claim 44, wherein the polypeptide of interest includes corestreptavidin and whereas said heterologous multimerization domaincomprises a biotin moiety or a Strep-tag.
 60. The method of claim 44,wherein the polypeptide of interest includes a biotin moiety or aStrep-tag and whereas said heterologous multimerization domain comprisescore streptavidin.
 61. The method of claim 44, wherein the polypeptideof interest and said heterologous multimerization domain are interlinkedvia a molecular linker.
 62. The method of claim 61, wherein at least oneof said heterologous multimerization domain and said molecular linkerinclude a hydrophilic region, said hydrophilic region being forfacilitating crystallization of the polypeptide of interest.
 63. Themethod of claim 61, wherein at least one of said heterologousmultimerization domain and said molecular linker include aconformationally rigid region, said conformationally rigid region beingfor facilitating crystallization of the polypeptide of interest.
 64. Themethod of claim 61, wherein at least one of said heterologousmultimerization domain and said molecular linker is selected so as todefine the spatial positioning and orientation of polypeptides of thepolypeptide of interest within said crystallizable molecular complex,thereby facilitating crystallization of the polypeptide of interest. 65.The method of claim 61, wherein said at least one molecular linkerincludes a region being capable of functioning as a purification tag,said purification tag being capable of facilitating purification of saidcrystallizable molecular complex, and/or of facilitating saidhomomultimerization of the polypeptide of interest.
 66. The method ofclaim 65, wherein said region being capable of functioning as apurification tag is selected from the group consisting of a T7 tag, ahistidine tag, a Strep-tag, core streptavidin, and biotin.
 67. Themethod of claim 44, wherein the polypeptide of interest includes aregion being capable of functioning as a purification tag, saidpurification tag being capable of facilitating purification of saidcrystallizable molecular complex, and/or of facilitating saidhomomultimerization of the polypeptide of interest.
 68. The method ofclaim 67, wherein said region being capable of functioning as apurification tag is selected from the group consisting of a T7 tag, ahistidine tag, a Strep-tag, core streptavidin, and biotin.
 69. Themethod of claim 44, wherein said molecule includes a metal-bindingmoiety capable of specifically binding a metal atom, said metal atombeing capable of facilitating crystallographic analysis of the crystal.70. The method of claim 69, wherein said metal-binding moiety is a metalbinding protein.
 71. The method of claim 70, wherein said metal bindingprotein is metallothionein.
 72. The method of claim 44, wherein thepolypeptide of interest is a membrane protein.
 73. The method of claim72, wherein said membrane protein is a G protein coupled receptor. 74.The method of claim 73, wherein said G protein coupled receptor isrhodopsin or is a class A G protein coupled receptor.
 75. The method ofclaim 74, wherein said class A G protein coupled receptor is m2muscarinic cholinergic receptor.
 76. The method of claim 44, wherein thepolypeptide of interest includes a metal-binding moiety capable ofspecifically binding a metal atom, said metal atom being capable offacilitating crystallographic analysis of the crystal.
 77. The method ofclaim 70, wherein said metal binding moiety is metallothionein.
 78. Acomposition-of-matter comprising at least two molecules of amolecule-of-interest interlinked via a heterologous molecular linker,wherein said heterologous molecular linker is selected so as to definethe relative spatial positioning and orientation of said at least twomolecules within the composition-of-matter, thereby facilitatingformation of a crystal therefrom under crystallization-inducingconditions.
 79. The composition-of-matter of claim 78, wherein themolecule-of-interest is a polypeptide.
 80. The composition-of-matter ofclaim 79, wherein said polypeptide is a membrane protein.
 81. Thecomposition-of-matter of claim 80, wherein said membrane protein is a Gprotein coupled receptor.
 82. The composition-of-matter of claim 81,wherein said G protein coupled receptor is rhodopsin or is a class A Gprotein coupled receptor.
 83. The composition-of-matter of claim 82,wherein said class A G protein coupled receptor is m2 muscariniccholinergic receptor.
 84. The composition-of-matter of claim 78, whereinsaid heterologous molecular linker includes at least one region capableof specifically binding said molecule-of-interest.
 85. Thecomposition-of-matter of claim 84, wherein said molecule-of-interest isa G protein coupled receptor and whereas said at least one regioncapable of specifically binding said molecule-of-interest is a moleculeselected from the group consisting of at least a portion of an arrestinmolecule, at least a portion of an arrestin molecule having a mutationat an amino acid residue position corresponding to position 90 in bovinevisual arrestin, at least a portion of an arrestin molecule having amutation at an amino acid residue position corresponding to position 175in bovine visual arrestin, SEQ ID NO: 3, and SEQ ID NO:
 4. 86. Thecomposition-of-matter of claim 85, wherein said at least a portion of anarrestin molecule is homologous to amino acid residues 11 to 190, or 11to 370 of human beta-arrestin-1a.
 87. The composition-of-matter of claim86, wherein said at least a portion of an arrestin molecule comprises aG protein coupled receptor-binding domain of said arrestin molecule. 88.The composition-of-matter of claim 85, wherein said mutation at an aminoacid residue position corresponding to position 90 in bovine visualarrestin is a mutation to a serine or threonine residue.
 89. Thecomposition-of-matter of claim 85, wherein said mutation at an aminoacid residue position corresponding to position 175 in bovine visualarrestin is a mutation to a glutamic acid or an asparagine residue. 90.The composition-of-matter of claim 85, wherein said G protein coupledreceptor is rhodopsin or is a class A G protein coupled receptor. 91.The composition-of-matter of claim 90, wherein said class A G proteincoupled receptor is m2 muscarinic cholinergic receptor.
 92. Thecomposition-of-matter of claim 78, wherein said heterologous molecularlinker includes a molecule selected from the group consisting of apolycyclic molecule, a polydentate ligand, a macrobicyclic cryptand, apolypeptide and a metal.
 93. The composition-of-matter of claim 78,wherein said molecule-of-interest is a G protein coupled receptor andwhereas said heterologous molecular linker comprises a molecule selectedfrom the group consisting of at least a portion of an arrestin molecule,at least a portion of an arrestin molecule having a mutation at an aminoacid residue position corresponding to position 90 in bovine visualarrestin, at least a portion of an arrestin molecule having a mutationat an amino acid residue position corresponding to position 175 inbovine visual arrestin, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, andSEQ ID NO:
 6. 94. The composition-of-matter of claim 93, wherein said atleast a portion of an arrestin molecule is homologous to amino acidresidues 11 to 190, or 11 to 370 of human beta-arrestin-1a.
 95. Thecomposition-of-matter of claim 94, wherein said at least a portion of anarrestin molecule comprises a G protein coupled receptor-binding domainof said arrestin molecule.
 96. The composition-of-matter of claim 93,wherein said mutation at an amino acid residue position corresponding toposition 90 in bovine visual arrestin is a mutation to a serine orthreonine residue.
 97. The composition-of-matter of claim 93, whereinsaid mutation at an amino acid residue position corresponding toposition 175 in bovine visual arrestin is a mutation to a glutamic acidor an asparagine residue.
 98. The composition-of-matter of claim 93,wherein said G protein coupled receptor is rhodopsin or is a class A Gprotein coupled receptor.
 99. The composition-of-matter of claim 98,wherein said class A G protein coupled receptor is m2 muscariniccholinergic receptor.
 100. The composition-of-matter of claim 78,wherein said heterologous molecular linker comprises core streptavidin.101. The composition-of-matter of claim 78, wherein said heterologousmolecular linker includes at least two non-covalently bound subunits.102. The composition-of-matter of claim 78, wherein said heterologousmolecular linker includes a hydrophilic region, said hydrophilic regionbeing for facilitating crystallization of said molecule-of-interest.103. The composition-of-matter of claim 78, wherein said heterologousmolecular linker includes a conformationally rigid region, saidconformationally rigid region being for facilitating crystallization ofsaid molecule-of-interest.
 104. The composition-of-matter of claim 78,wherein said heterologous molecular linker is selected such that thecomposition-of-matter is capable of generating a crystal selected fromthe group consisting of a 2D crystal, a helical crystal and a 3Dcrystal.
 105. The composition-of-matter of claim 78, wherein saidheterologous molecular linker includes a metal-binding moiety capable ofspecifically binding a metal atom, said metal atom being capable offacilitating crystallographic analysis of the crystal.
 106. Thecomposition-of-matter of claim 105, wherein said metal-binding moiety isa metal-binding protein.
 107. The composition-of-matter of claim 106,wherein said metal binding protein is metallothionein.
 108. Thecomposition-of-matter of claim 78, wherein said heterologous molecularlinker includes a region being capable of functioning as a purificationtag, said purification tag being capable of facilitating purification ofthe crystallizable composition-of-matter, and/or of facilitating saidinterlinking of said at least two molecules of a molecule-of-interest.109. The composition-of-matter of claim 78, wherein said region beingcapable of functioning as a purification tag is selected from the groupconsisting of a T7 tag, a histidine tag, a Strep-tag, core streptavidin,and biotin.
 110. The composition-of-matter of claim 78, wherein saidmolecule-of-interest includes a region being capable of functioning as apurification tag, said purification tag being capable of facilitatingpurification of the composition-of-matter, and/or of facilitating saidinterlinking of said at least two molecules of a molecule-of-interest.111. The composition-of-matter of claim 110, wherein said region beingcapable of functioning as a purification tag is selected from the groupconsisting of a T7 tag, a histidine tag, a Strep-tag, core streptavidin,and biotin.
 112. The composition-of-matter of claim 78, wherein saidmolecule-of-interest includes a metal-binding moiety capable ofspecifically binding a metal atom, said metal atom being capable offacilitating crystallographic analysis of the crystal.
 113. Thecomposition-of-matter of claim 112, wherein said metal-binding moiety isa metal binding protein.
 114. The composition-of-matter of claim 113,wherein said metal-binding protein is metallothionein.
 115. A nucleicacid construct comprising a polynucleotide segment encoding a chimericpolypeptide including: (a) a first polypeptide region being capable ofspecifically binding a molecule-of-interest; and (b) a secondpolypeptide region being capable of specifically binding a metal atom.116. The nucleic acid construct of claim 115, wherein saidmolecule-of-interest is a G protein coupled receptor and whereas saidchimeric polypeptide comprises SEQ ID NO: 5 or SEQ ID NO:
 6. 117. Thenucleic acid construct of claim 116, wherein said G protein coupledreceptor is rhodopsin or is a class A G protein coupled receptor. 118.The nucleic acid construct of claim 117, wherein said class A G proteincoupled receptor is m2 muscarinic cholinergic receptor.
 119. The nucleicacid construct of claim 115, wherein said molecule-of-interest is a Gprotein coupled receptor and whereas said first polypeptide regioncomprises a molecule selected from the group consisting of at least aportion of an arrestin molecule, at least a portion of an arrestinmolecule having a mutation at an amino acid residue positioncorresponding to position 90 in bovine visual arrestin, at least aportion of an arrestin molecule having a mutation at an amino acidresidue position corresponding to position 175 in bovine visualarrestin, SEQ ID NO: 3, and SEQ ID NO:
 4. 120. The nucleic acidconstruct of claim 119, wherein said at least a portion of an arrestinmolecule is homologous to amino acid residues 11 to 190, or 11 to 370 ofhuman beta-arrestin-1a.
 121. The nucleic acid construct of claim 120,wherein said at least a portion of an arrestin molecule comprises a Gprotein coupled receptor-binding domain of said arrestin molecule. 122.The nucleic acid construct of claim 119, wherein said mutation at anamino acid residue position corresponding to position 90 in bovinevisual arrestin is a mutation to a serine or threonine residue.
 123. Thenucleic acid construct of claim 119, wherein said mutation at an aminoacid residue position corresponding to position 175 in bovine visualarrestin is a mutation to a glutamic acid or an asparagine residue. 124.The nucleic acid construct of claim 119, wherein said G protein coupledreceptor is rhodopsin or is a class A G protein coupled receptor. 125.The nucleic acid construct of claim 124, wherein said class A G proteincoupled receptor is m2 muscarinic cholinergic receptor.
 126. The nucleicacid construct of claim 115, wherein the molecule-of-interest is apolypeptide.
 127. The nucleic acid construct of claim 126, wherein saidpolypeptide is a membrane protein.
 128. The nucleic acid construct ofclaim 127, wherein said membrane protein is a G protein coupledreceptor.
 129. The nucleic acid construct of claim 128, wherein said Gprotein coupled receptor is rhodopsin or is a class A G protein coupledreceptor.
 130. The nucleic acid construct of claim 129, wherein saidclass A G protein coupled receptor is m2 muscarinic cholinergicreceptor.
 131. The nucleic acid construct of claim 115, wherein saidsecond polypeptide region is metallothionein.
 132. The nucleic acidconstruct of claim 115, wherein said chimeric polypeptide is selectedsuch that when combined with molecules of said molecule-of-interestunder suitable conditions, said chimeric polypeptide and said moleculesform a crystallizable molecular complex which is capable of forming acrystal containing said molecule-of-interest when subjected tocrystallization-inducing conditions.
 133. The nucleic acid construct ofclaim 115, wherein said chimeric polypeptide is selected such that whencombined with molecules of said molecule-of-interest and said metal atomunder suitable conditions, said chimeric polypeptide and said moleculesform a crystallizable molecular complex which is capable of forming acrystal containing said molecule-of-interest when subjected tocrystallization-inducing conditions.
 134. The nucleic acid construct ofclaim 132, wherein said metal atom facilitates crystallographic analysisof said crystal.
 135. The nucleic acid construct of claim 132, whereinsaid chimeric polypeptide includes a hydrophilic region, saidhydrophilic region being for facilitating crystallization of saidmolecule-of-interest.
 136. The nucleic acid construct of claim 132,wherein said chimeric polypeptide includes a conformationally rigidregion, said conformationally rigid region being for facilitatingcrystallization of said molecule-of-interest.
 137. The nucleic acidconstruct of claim 132, wherein said chimeric polypeptide is selected soas to define the spatial positioning and orientation of saidmolecule-of-interest within said crystallizable molecular complex,thereby facilitating crystallization of said molecule-of-interest. 138.The nucleic acid construct of claim 132, wherein said chimericpolypeptide is selected such that said crystallizable molecular complexformed is capable of generating a crystal selected from the groupconsisting of a 2D crystal, a helical crystal and a 3D crystal.
 139. Thenucleic acid construct of claim 132, wherein said chimeric polypeptidefurther includes a polypeptide region being capable of functioning as apurification tag, said purification tag being capable of facilitatingpurification of said crystallizable molecular complex, and/or offacilitating said binding of a molecule-of-interest.
 140. The nucleicacid construct of claim 139, wherein said region being capable offunctioning as a purification tag is selected from the group consistingof a T7 tag, a histidine tag, a Strep-tag, core streptavidin. andbiotin.
 141. A nucleic acid construct comprising a polynucleotidesegment encoding a chimeric polypeptide including: (a) a firstpolypeptide region being capable of specifically binding amolecule-of-interest; (b) a second polypeptide region being capable ofhomomultimerization into a complex of defined geometry; and (c) a thirdpolypeptide region being capable of specifically binding a metal atom.142. The nucleic acid construct of claim 141, wherein saidmolecule-of-interest is a G protein coupled receptor and whereas saidfirst polypeptide region is selected from the group consisting of atleast a portion of an arrestin molecule, at least a portion of anarrestin molecule having a mutation at an amino acid residue positioncorresponding to position 90 in bovine visual arrestin, at least aportion of an arrestin molecule having a mutation at an amino acidresidue position corresponding to position 175 in bovine visualarrestin, SEQ ID NO: 3, and SEQ ID NO:
 4. 143. The nucleic acidconstruct of claim 142, wherein said at least a portion of an arrestinmolecule is homologous to amino acid residues 11 to 190, or 11 to 370 ofhuman beta-arrestin-1a.
 144. The nucleic acid construct of claim 143,wherein said at least a portion of an arrestin molecule comprises a Gprotein coupled receptor-binding domain of said arrestin molecule. 145.The nucleic acid construct of claim 142, wherein said mutation at anamino acid residue position corresponding to position 90 in bovinevisual arrestin is a mutation to a serine or threonine residue.
 146. Thenucleic acid construct of claim 9, wherein said mutation at an aminoacid residue position corresponding to position 175 in bovine visualarrestin is a mutation to a glutamic acid or an asparagine residue. 147.The nucleic acid construct of claim 142, wherein said G protein coupledreceptor is rhodopsin or is a class A G protein coupled receptor. 148.The nucleic acid construct of claim 147, wherein said class A G proteincoupled receptor is m2 muscarinic cholinergic receptor.
 149. The nucleicacid construct of claim 141, wherein said second polypeptide regioncomprises core streptavidin.
 150. The nucleic acid construct of claim141, wherein said molecule-of-interest is a G protein coupled receptorand whereas said chimeric polypeptide comprises SEQ ID NO: 5 or SEQ IDNO:
 6. 151. The nucleic acid construct of claim 150, wherein said Gprotein coupled receptor is rhodopsin or is a class A G protein coupledreceptor.
 152. The nucleic acid construct of claim 151, wherein saidclass A G protein coupled receptor is m2 muscarinic cholinergicreceptor.
 153. The nucleic acid construct of claim 141, wherein saidthird polypeptide region comprises metallothionein.
 154. The nucleicacid construct of claim 141, wherein the molecule-of-interest is apolypeptide.
 155. The nucleic acid construct of claim 154, wherein saidpolypeptide is a membrane protein.
 156. The nucleic acid construct ofclaim 155, wherein said membrane protein is a G protein coupledreceptor.
 157. The nucleic acid construct of claim 156, wherein said Gprotein coupled receptor is rhodopsin or is a class A G protein coupledreceptor.
 158. The nucleic acid construct of claim 157, wherein saidclass A G protein coupled receptor is m2 muscarinic cholinergicreceptor.
 159. The nucleic acid construct of claim 141, wherein saidchimeric polypeptide is selected such that when combined with moleculesof said molecule-of-interest, said chimeric polypeptide and saidmolecules form a crystallizable molecular complex of defined geometrywhich is capable of forming a crystal containing saidmolecule-of-interest when subjected to crystallization-inducingconditions.
 160. The nucleic acid construct of claim 159, wherein saidchimeric polypeptide includes a hydrophilic region, said hydrophilicregion being for facilitating crystallization of saidmolecule-of-interest.
 161. The nucleic acid construct of claim 159,wherein said chimeric polypeptide includes a conformationally rigidregion, said conformationally rigid region being for facilitatingcrystallization of said molecule-of-interest.
 162. The nucleic acidconstruct of claim 159, wherein said chimeric polypeptide is selected soas to define the spatial positioning and orientation of molecules ofsaid molecule-of-interest within said crystallizable molecular complex,thereby facilitating crystallization of said molecule-of-interest. 163.The nucleic acid construct of claim 159, wherein said chimericpolypeptide is selected such that said crystallizable molecular complexof defined geometry formed is capable of generating a crystal selectedfrom the group consisting of a 2D crystal, a helical crystal and a 3Dcrystal.
 164. The nucleic acid construct of claim 159, wherein saidmetal atom facilitates crystallographic analysis of saidmolecule-of-interest contained in said crystal.
 165. The nucleic acidconstruct of claim 159, wherein said chimeric polypeptide furtherincludes a polypeptide region being capable of functioning as apurification tag, said purification tag being capable of facilitatingpurification of said crystallizable molecular complex, and/or offacilitating said binding of a molecule-of-interest.
 166. The nucleicacid construct of claim 165, wherein said region being capable offunctioning as a purification tag is selected from the group consistingof a T7 tag, a histidine tag, a Strep-tag, and core streptavidin.
 167. Amethod of purifying a G protein coupled receptor from a samplecontaining the G protein coupled receptor, the method comprisingsubjecting the sample to affinity chromatography using an affinityligand selected from the group consisting of at least a portion of anarrestin molecule, at least a portion of an arrestin molecule having amutation at an amino acid residue position corresponding to position 90in bovine visual arrestin, at least a portion of an arrestin moleculehaving a mutation at an amino acid residue position corresponding toposition 175 in bovine visual arrestin, a molecule defined by SEQ ID NO:3, and a molecule defined by SEQ ID NO: 4, thereby purifying the Gprotein coupled receptor.
 168. The method of claim 167, wherein said atleast a portion of an arrestin molecule is homologous to amino acidresidues 11 to 190, or 11 to 370 of human beta-arrestin-1a.
 169. Themethod of claim 168, wherein said at least a portion of an arrestinmolecule comprises a G protein coupled receptor-binding domain of saidarrestin molecule.
 170. The method of claim 167, wherein said mutationat an amino acid residue position corresponding to position 90 in bovinevisual arrestin is a mutation to a serine or threonine residue.
 171. Themethod of claim 167, wherein said mutation at an amino acid residueposition corresponding to position 175 in bovine visual arrestin is amutation to a glutamic acid or an asparagine residue.
 172. The method ofclaim 167, wherein said G protein coupled receptor is rhodopsin or is aclass A G protein coupled receptor.
 173. The method of claim 172,wherein said class A G protein coupled receptor is m2 muscariniccholinergic receptor.
 174. The method of claim 167, wherein saidaffinity ligand includes a region being capable of functioning as apurification tag, said purification tag being capable of facilitatingattachment of said affinity ligand to an affinity chromatography matrix.175. The method of claim 174, wherein said region being capable offunctioning as a purification tag is selected from the group consistingof a T7 tag, a histidine tag, a Strep-tag, core streptavidin, andbiotin.